Heteromultivalent particle compositions

ABSTRACT

A composition for use in diagnostic and therapeutic applications includes a heteromultivalent nanoparticle or microparticle having an outer surface and a plurality of targeting moieties conjugated to the surface of the nanoparticle or microparticle, the targeting moieties includes a first activated platelet targeting moiety and a second activated platelet targeting moiety.

RELATED APPLICATION

This application is continuation in part of U.S. patent application Ser.No. 13/863,005 filed Apr. 15, 2013, now U.S. Pat. No. 9,107,963, whichclaims priority from U.S. Provisional Application No. 61/623,607 filedApr. 13, 2012, the subject matter of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This application relates to heteromultivalent nanoparticle ormicroparticle compositions selective for activated platelets and to theuse of the heteromultivalent nanoparticle or microparticle compositionsfor diagnostic and therapeutic applications.

BACKGROUND

Vascular diseases, leading to thrombo-occlusive and ischemic end points,are the leading cause of tissue morbidity and mortality in the UnitedStates and globally. Current treatments for vascular disease includeboth endovascular and pharmacotherapy strategies. For example,angioplasty/stenting, which is invasive, can cause restenosis andsecondary thrombotic events. In addition, bypass grafting is bothinvasive and can lead to graft failure. In vascular diseasemanifestations, such as atherosclerotic plaque progression and ruptureas well as events leading to thrombosis and restenosis, diseasesite-selective delivery of therapeutic agents (e.g., thrombolytic oranti-proliferative drugs) and diagnostic probes (e.g., MRI or CTcontrast agents) can provide significantly enhanced treatment efficacycompared to systemic administration of the same agents. This is because,in direct systemic administration, a significant fraction of the agentsmay get cleared rapidly or may get deactivated by plasma action, therebyreducing their therapeutic concentration at the target disease site.Moreover, systemic distribution of the drug can cause unwanted sideeffects in un-involved tissues, such as systemic coagulopathic andhemorrhagic effects. Thus, methods allowing for disease site-selectivedelivery can have a significant clinical benefit.

SUMMARY

Embodiments described herein relate to a heteromultivalent nanoparticleor microparticle composition for use in diagnostic and therapeuticapplications. The composition includes a heteromultivalent nanoparticleor microparticle having an outer surface and a plurality of targetingmoieties conjugated to the surface of the nanoparticle or microparticle.The targeting moieties can include a first activated platelet targetingmoiety and a second activated platelet targeting moiety. The compositionis capable of binding activated platelets at a thrombus site under ahemodynamic shear environment. In some embodiments, the nanoparticle ormicroparticle can include a liposome.

In some embodiments, the first activated platelet targeting moietyincludes a GPIIb-IIIa-binding peptide and the second activated platelettargeting moiety includes a p-selectin binding peptide. TheGPIIb-IIIa-binding peptide can include a RGD small peptide and thep-selectin binding peptide can include a small peptide having the aminoacid sequence EWVDV (SEQ ID NO:1). In some embodiments, the RGD smallpeptide has an amino acid sequence of SEQ ID NO:3, and the p-selectinbinding peptide has the amino acid sequence of SEQ ID NO:5. The ratio ofGPIIb-IIIa-binding peptide to p-selectin binding peptide provided on thenanoparticle or microparticle surface can be about 80:20 to about 20:80.The GPIIb-IIIa-binding peptide and p-selectin binding peptide providedon the nanoparticle or microparticle surface can have a total mol % ofabout 5 to about 20 with respect to total lipid content.

The first and second activated platelet targeting moieties can beconjugated to the nanoparticle or microparticle surface with PEGlinkers. The first and second activated platelet targeting moieties canbe spatially or topographically arranged on the nanoparticle ormicroparticle surface such that the first and second activated platelettargeting moieties do not spatially mask each other and the nanoparticleor microparticle is able to bind to an activated platelet with exposedactivated platelet receptors thereby enhancing retention of thenanoparticle or microparticle construct onto activated platelets underhemodynamic flow.

In some embodiments, the composition further includes a therapeuticand/or imaging agent. The agent can be encapsulated within thenanoparticle or microparticle construct. The therapeutic agent can beselected from the group consisting of an anti-thrombotic agent and athrombolytic agent. The therapeutic agent can also include tissueplasminogen activator (tPA). In some embodiments, the nanoparticle ormicroparticle further includes Golden Nanorods (GNRs) conjugated to thesurface, the GNRs can allow photothermal destabilization of thenanoparticle or microparticle construct and therapeutic and/or imagingagent release in response to near-infrared (NIR) light.

The application also relates to a method of delivering a therapeuticand/or imaging agent to activated platelets in a subject. The methodincludes administering to the subject a composition. The compositionincludes a therapeutic agent. The composition also includesheteromultivalent nanoparticle or microparticle having an outer surfaceand a plurality of targeting moieties conjugated to the surface of thenanoparticle or microparticle. The targeting moieties can include afirst activated platelet targeting moiety and a second activatedplatelet targeting moiety. The composition is capable of bindingactivated platelets at a thrombus site under a hemodynamic shearenvironment. In some embodiments, the nanoparticle or microparticle caninclude a liposome.

In some embodiments, the first activated platelet targeting moietyincludes a GPIIb-IIIa-binding peptide and the second activated platelettargeting moiety includes a p-selectin binding peptide. TheGPIIb-IIIa-binding peptide can include a RGD small peptide, and thep-selectin binding peptide can include a small peptide having the aminoacid sequence EWVDV (SEQ ID NO:1). In some embodiments, the RGD smallpeptide has an amino acid sequence of SEQ ID NO:3, and the p-selectinbinding peptide has the amino acid SEQ ID NO:5. The ratio ofGPIIb-IIIa-binding peptide to p-selectin binding peptide provided on thenanoparticle or microparticle surface can be about 80:20 to about 20:80.The GPIIb-IIIa-binding peptide and p-selectin binding peptide providedon the nanoparticle or microparticle surface can have a total mol % ofabout 5 to about 20 with respect to total lipid content.

The first and second activated platelet targeting moieties can beconjugated to the nanoparticle or microparticle surface with PEGlinkers. The first and second activated platelet targeting moieties canbe spatially or topographically arranged on the nanoparticle ormicroparticle surface such that the first and second activated platelettargeting moieties do not spatially mask each other and the nanoparticleor microparticle is able to bind to an activated platelet with exposedactivated platelet receptors thereby enhancing retention of thenanoparticle or microparticle onto activated platelets under hemodynamicflow.

The therapeutic agent can be selected from the group consisting of ananti-thrombotic agent and a thrombolytic agent. The therapeutic agentcan also include tissue plasminogen activator (tPA). In someembodiments, the nanoparticle or microparticle further includes GoldenNanorods (GNRs) conjugated to the surface. The GNRs can allowphotothermal destabilization of the nanoparticle or microparticleconstruct and therapeutic and/or imaging agent release in response tonear-infrared (NIR) light.

The application further relates to a method of treating an occlusivevascular disease in a subject. The method includes administering to thesubject a composition. The composition includes a therapeutic agent. Thecomposition also includes heteromultivalent nanoparticle ormicroparticle having an outer surface and a plurality of targetingmoieties conjugated to the surface of the nanoparticle or microparticle.The targeting moieties include a first activated platelet targetingmoiety and a second activated platelet targeting moiety. The compositionis capable of binding activated platelets at a thrombus site under ahemodynamic shear environment.

In some embodiments, the nanoparticle or microparticle can furtherincludes Golden Nanorods (GNRs) conjugated to the surface, the GNRsallowing photothermal destabilization of the nanoparticle ormicroparticle construct and therapeutic and/or imaging agent release inresponse to near-infrared (NIR) light.

In some embodiments, the occlusive vascular disease is selected from thegroup consisting of stroke, myocardial infarction, peripheral arterialdiseases and deep vein thrombosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing the design of liposomalconstructs surface-functionalized with two types of ligands forsimultaneous targeting of GPIIb-IIIa integrin and P-selectin expressedat high quantity on the membrane surface of activated platelets involvedin thrombotic and inflammatory events in vascular disease.

FIG. 2 illustrates a process for fabricating the heteromultivalentliposomal constructs for dual targeting of activated platelets by RGDpeptide (SEQ ID NO: 2) and DAEWVDVS (SEQ ID NO:5); bottom panel showsrepresentative DLS characterization data for the constructs before andafter extrusion; after extrusion the constructs are largely monodispersewith an average diameter of 150 nm.

FIG. 3(A-E) illustrate a fluorescence microscopy experiment to establishthe specific binding of RGD-modified liposomes to activated platelets;top panel shows the experiment procedure; middle panel showsrepresentative microscope images of control and test conditions (Athrough E); the quantitative analysis of fluorescence intensity frommultiple images (n=10 per test or control condition) at these conditionsare shown in the graph at the bottom, confirming the enhanced binding ofactivated platelets by RGD-modified liposomes compared to unmodifiedliposomes.

FIG. 4(A-E) illustrates a fluorescence microscopy experiment toestablish the specific binding of DAEWVDVS (SEQ ID NO: 5)-modifiedliposomes to activated platelets; top panel shows the experimentprocedure with EWVDVS (SEQ ID NO: 1); middle panel shows representativemicroscope images of control and test conditions (A through E); thequantitative analysis of fluorescence intensity from multiple images(n=10 per test or control condition) at these conditions are shown inthe graph at the bottom, confirming the enhanced binding of activatedplatelets by DAEWVDVS (SEQ ID NO: 5)-modified liposomes compared tounmodified liposomes.

FIG. 5(A-B) illustrate the specific receptor blocking studies analyzedby fluorescence microscopy to establish that RGD-modified liposomes bindto GPIIb-IIIa on activated platelets; top panel shows the experimentprocedure; middle panel shows representative microscope images ofcontrol and test conditions; the quantitative analysis of fluorescenceintensity from multiple images (n=10 per test or control condition) atthese conditions are shown in the graph at the bottom, confirming thatpre incubation of activated platelets with RGD-modified liposomesresults in significant blocking of fluorescent antibody specificallybinding to activated platelet GPIIb-IIIa; this result in combinationwith results shown in FIG. 3 confirm that the RGD-modified liposomes canspecifically target and bind to GPIIb-IIIa on activated platelets.

FIG. 6(A-B) illustrate specific receptor blocking studies analyzed byfluorescence microscopy to establish that DAEWVDVS (SEQ ID NO:5)-modified liposomes bind to P-selectins on activated platelets; toppanel shows the experiment procedure; middle panel shows representativemicroscope images of control and test conditions; the quantitativeanalysis of fluorescence intensity from multiple images (n=10 per testor control condition) at these conditions are shown in the graph at thebottom, confirming that pre-incubation of activated platelets withDAEWVDVS (SEQ ID NO: 5)-modified liposomes results in significantblocking of fluorescent antibody specifically binding to activatedplatelet P-selectins; this result in combination with results shown inFIG. 4 confirm that the DAEWVDVS (SEQ ID NO: 5)-modified liposomes canspecifically target and bind to P-selectins on activated platelets.

FIG. 7 illustrates schematic representation of experimental set-up andprocedure for the PPFC experiments to establish the enhanced binding andretention of peptide-modified liposomes to activated platelets underhemodynamic flow relevant shear stress ranges over time; the bottom leftalso shows representative SEM images of albumin-coated surface area andcollagen-coated surface area after incubation with activated platelets,confirming that the collagen-coated surface has a significantly highdensity of active platelets; allowing the test and control liposomes tointeract with platelet-rich (collagen-coated) and platelet-deficient(albumin-coated) surface regions on the same slide under various shearstress ranges in the PPFC set-up for effective analysis of liposomebinding and retention.

FIG. 8(A-B) illustrate a representative flow cytometry results showing(A) the gated activated platelet population in whole blood aliquotsunder analysis and (B) the fluorescence histograms from plateletpopulation interacting with unmodified (non-targeted), RGD-(SEQ ID NO:2) or DAEWVDVS (SEQ ID NO: 5)-modified (singly-targeted) andsimultaneous RGD-(SEQ ID NO: 2) and DAEWVDVS (SEQ ID NO: 5)-modified(dual-targeted) fluorescently-labeled liposomes; it is evident thatthough the singly-targeted liposomes are capable of binding activatedplatelets significantly more than the non-targeted liposomes, thedual-targeted liposomes have even higher extent of binding activatedplatelets when compared to the singly-targeted liposomes.

FIG. 9 illustrates a representative fluorescence microscopy images andquantitative data from PPFC experiments to study binding and retentionof test (singly or dual-targeted) and control (non-targeted) liposomesto activated platelet-coated surface versus platelet-deficient surfaceunder flow at three different shear stress values (low-to-high shear)over a period of 45 min (30 min liposomal suspension flow+15 min 1×PBSflow); it is evident that in a dynamic flow environment, dual-targetedliposomes are capable of binding and staying retained on target cells(activated platelets) at significantly enhanced levels over time at allshear stress values compared to non-targeted and even singly-targetedliposomes.

FIG. 10(A-C) are graphical illustrations of nanovehicle binding toactive platelets using varying mole % of ligands directed to GPIIb-IIIaand P-selectin at a flow rate of (A) 5 Dynes/cm², (B) 25 Dynes/cm² and(C) 55 Dynes/cm². Binding is measured by the surface averagedfluorescence intensity (AU) over time (mins). Results show the liposomebinding generated at flow rates of 5 Dynes/cm², 25 Dynes/cm² and 55Dynes/cm² and using 2.5%, 5%, and 10% DAEWVDVS (SEQ ID NO: 5) peptidedirected to P-selectin (singly-targeted) and 5% DAEWVDVS (SEQ ID NO: 5)peptide/RGD (SEQ ID NO: 2) heteromultivalent peptides.

FIG. 11(A-C) are graphical illustrations of nanovehicle binding toactive platelets using fixed mole % but varying ratios of ligandsdirected to GPIIb-IIIa and P-selectin at flow rates of (A) 5 Dynes/cm²,(B) 25 Dynes/cm² and (C) 55 Dynes/cm². Binding is measured by thesurface averaged fluorescence intensity (AU) over time (mins). Resultsshow the thrombus-targeted liposome binding generated at a flow rate of5 Dynes/cm² using 2.5%, 5%, and 10% DAEWVDVS (SEQ ID NO: 5) peptidedirected to P-selectin (singly-targeted) and 5% DAEWVDVS (SEQ ID NO: 5)peptide/RGD heteromultivalent peptides.

FIG. 12 is a schematic illustration showing the design of liposomalconstructs surface-functionalized with two types of ligands (SEQ ID NO:1 and SEQ ID NO: 3) for simultaneous targeting of GPIIb-IIIa integrinand P-selectin expressed at high quantity on the membrane surface ofactivated platelets involved in thrombotic and inflammatory events invascular disease and further surface functionalized with Gold Nanorods(GNRs) allowing for the NIR-triggered release of a drug from theliposomal construct.

FIG. 13 illustrates a schematic representation of the fabrication ofGNR-decorated liposomes in accordance with an embodiment of theapplication.

FIG. 14 shows a representative TEM and absorption spectra for GNRsfabricated using the seed-mediated method.

FIG. 15 illustrates Single Photon Emission Computed Tomography (SPECT)images confirming enhanced thrombus-selective accumulation ofplatelet-targeted liposomes in carotid injury.

FIG. 16 illustrates a schematic representation of liposomessurface-modified with fibrinogen-mimetic peptides for targetingintegrins GPIIb-IIIa on activated platelets at a site of vascular injuryin accordance with an embodiment of the present application.

FIG. 17(A-C) illustrate the amino acid sequence structure andcorresponding mass spectral (MALDI-TOF) data for plateletGPIIb-IIIa-targeted (a) linear RGD), (b) cyclic RGD and (c) lipopeptideconjugate DSPE-PEG-cRGD.

FIG. 18 illustrates the IC₅₀ estimation by aggregometry assay for freelRGD and cRGD shows lower IC₅₀ for cRGD by-2 orders of magnitude,compared to lRGD.

FIG. 19(A-D) are scanning electron microscopy (SEM) images confirmingthe presence of activated platelet monolayer on collagen-coated glasscoverslip surface (A); co-incubation of active platelets withAlexaFluor-546-labeled fibrinogen and NBD-labeled peptide-modifiedliposomes resulted in simultaneous platelet-binding of both, and henceenabled dual fluorescence imaging of the same field of view; comparisonof fluorescence images showed similar level of AlexaFluor-546fluorescence for lRGD-(B1) and cRGD-liposome (C1) incubated plateletssuggesting similar level of fibrinogen binding; NBD-fluorescence,however, was significantly enhanced for cRGD-liposome incubated sample(C2), compared to lRGD-liposome incubated sample (B2) as suggested bythe mean fluorescent intensity data (D); this indicates enhanced bindingof cyclic RGD-modified liposomes to activated platelets.

FIG. 20(A-C) illustrate histograms showing flow cytometry studies onplatelet-liposome interactions in vitro; platelet activation level wasconfirmed by co-labeling platelets in human whole blood aliquots withFITC-anti-CD41a (antibody to GPIIb-IIIa) and PE-antiCD62P (antibody toP-selectin), (A) before and (B) after addition of agonist ADP; FIG.20(C) is a graphical representation showing that incubation of activatedplatelets with NBD-labeled peptide-modified liposomes enhanced NBDfluorescence for cRGD-liposome incubated sample compared tolRGD-liposome incubated sample, over background (unlabeled).

FIG. 21(A-G) illustrate in vivo targeting of RGD-modified liposomes in arat carotid injury model; SEM image of (A) uninjured luminal surface ofcarotid artery shows characteristic spindle shaped endothelial cells andextracellular matrix fibers; following catheter-induced injury, SEMimage of the luminal surface shows (B) numerous adhered platelets inactivated state (B, inset, magnified); repurfusion of blood through theinjury site results in acute thrombotic and coagulatory event, asevidenced by (C) SEM image of the fibrin clot on the luminal surface;exposure of injured surface to NBD-labeled lRGE-, lRGD-, andcRGD-liposomes via an in vivo injection of respective liposomeformulation, results in enhanced binding of (G) cRGD-liposomes to theinjury site, comparted to (E) lRGE- and (F)lRGD-liposomes, as evidencedby ex-vivo fluorescence imaging; (D) shows the fluorescence image of theluminal surface without exposure to NBD-labeled liposomes, forreference.

FIG. 22 illustrates a schematic diagram of an exemplary method ofliposome preparation including a molecular model representation of thelipid bilayer membrane of the liposome, and the dynamic light scattering(DLS) analysis of liposome size distribution before and after extrusionthrough nanoporous membrane.

FIG. 23(A-F) are representative fluorescence and phase contrastmicroscopy images from in vitro studies of active platelet targeting bypeptide-modified fluorescently labeled liposomes; (A) RGEliposome-incubated sample showed minimal fluorescence, while (B)cRGD-liposome incubated sample showed significantly enhancedfluorescence; parallel analysis of cRGD liposome-incubated sample with(C) fluorescence and (D) phase contrast microscopy provided evidence forone-to-one correspondence of fluorescence spots with active platelets,thereby emphasizing that the liposome binding is platelet-specific; (E)represents the surface-averaged fluorescence intensity analysis of theRGE-liposome incubated and the cRGD-liposome incubated samples; (F)shows representative flow cytometry data of NBD fluorescence intensityhistogram overlay for platelets without any liposome incubation, withliposome incubation but without ADP pre-incubation (predominantlyinactive platelets) and with liposome incubation after ADPpre-incubation (predominantly active platelets), establishing that theNBD-labeled cRGD-liposomes have specific enhanced binding to activatedplatelets.

FIG. 24(A-D2) are representative images from SEM studies of activeplatelet targeting by peptide-modified Nanogold-labeled liposomes; SEManalysis of collagen-coated coverslip exposed to human plateletsuspension in presence of ADP showed (A) monolayer of adhered plateletswhich were in highly activated condition as evidenced by (B) highlyfolded membrane structure, ‘spread’ morphology and branched outpseudopods; SEM images in secondary electron mode of (C1)RGE-liposome-incubated platelet and (D1) cRGD-liposome-incubatedplatelet showed similar membrane morphology and vesicular structures,but corresponding images in backscatter mode showed (C2) minimal goldcontrast enhancement on the membrane of RGE-liposome-incubated platelet,while (D2) significant contrast enhancement of the membrane ofcRGD-liposome-incubated platelet; in addition, multiple bright sphericalvesicular structures (arrowheads in D2) were visible oncRGD-liposome-incubated platelet, which are possibly intact liposomesadhered on the membrane and pseudopods.

FIG. 25(A-L) are representative ex vivo images from studies of activeplatelet binding of peptide-modified fluorescently labeled liposomes invivo in a rat carotid injury restenosis model; (A) shows the schematicof the in vivo model used in the study; (B) and (D) show representativefluorescence and SEM images of the carotid luminal surface withoutinjury; (E) is an SEM image of the carotid luminal surface immediatelyafter frictional injury with inflated balloon catheter, showingsignificant wall damage and endothelial denudation; after 1 hr of bloodrepurfusion through such injured carotid, SEM image of excised sectionsshowed (F) numerous adhered platelets and upon magnification (G)arrested platelets in dense fibrin mesh were visible, signifying acutethrombotic environment; the presence of adhered platelets was confirmedby (C) fluorescence imaging after staining the injured section withFITC-tagged anti-rat CD42d (stains platelet GPV); introduction offluorescently labeled RGE-liposomes in the injured section after (H) 1hr and (J) 2 hr blood repurfusion showed only minimal staining ofplatelets; in contrast, introduction of fluorescently labeled cRGDliposomes showed significant platelet staining in both (I) 1 hr and (K)2 hr repurfusion situations; for 2 hr repurfusion sections, highlyfluorescent clumps of aggregated platelets were also visible; (L)statistical analysis of fluorescence intensity from images of liposomeexposed carotid sections for several batches of animals, confirmed theenhanced binding of cRGD-liposomes at the injury site.

FIG. 26(A1-B2) are representative ex vivo SEM images from studies ofactive platelet targeting by peptide modified Nanogold-labeled liposomesin vivo in a rat carotid injury restenosis model; the secondary electronmode images of both (A1) RGE-liposome-incubated and (B1)cRGD-liposome-incubated samples showed similar acute thromboticenvironment with platelets arrested in dense fibrin mesh, but thecorresponding backscatter image (A2) of RGE-liposome-incubated sampleshowed only minimal contrast enhancement while the backscatter image(B2) of cRGD-liposome-incubated sample showed substantial contrastenhancement of cellular structures arrested in the fibrin mesh,suggesting enhanced binding of Nanogold-labeled cRGD-liposomes toplatelets.

FIG. 27(A-C) illustrates selective binding of thrombus-targetedliposomes in accordance with an embodiment of the invention compared toplatelet-mimetic liposomes at varying ratios of ligands directed toGPIIb-IIIa (FMP) and P-selectin (SMP) and von Willebrand factor-bindingpeptides (VBPs) and collagen-binding peptides (CBPs) at a flow rate of(A) 5 Dynes/cm², (B) 25 Dynes/cm² and (C) 55 Dynes/cm².

DETAILED DESCRIPTION

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of theapplication.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “subject” can refer to any animal including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish (e.g., zebrafish)), non-human primates,ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, orcanines felines, aves, etc.).

The terms “diminishing,” “reducing,” or “preventing,” “inhibiting,” andvariations of these terms, as used herein include any measurabledecrease, including complete or substantially complete inhibition. Theterms “enhance” or “enhanced” as used herein include any measurableincrease or intensification.

As used herein, the term “small molecule” can refer to lipids,carbohydrates, polynucleotides, polypeptides, or any other organic orinorganic molecules.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues related naturally occurring structural variants, andsynthetic non-naturally occurring analogs thereof linked via peptidebonds or modified peptide bonds (i.e., peptide isosteres), relatednaturally occurring structural variants, and synthetic non-naturallyoccurring analogs thereof, glycosylated polypeptides, and all “mimetic”and “peptidomimetic” polypeptide forms. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term can refer to an oligopeptide, peptide, polypeptide, or proteinsequence, or to a fragment, portion, or subunit of any of these. Theterm “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

A “portion” of a polypeptide means at least about three sequential aminoacid residues of the polypeptide. It is understood that a portion of apolypeptide may include every amino acid residue of the polypeptide.

“Mutants,” “derivatives,” and “variants” of a polypeptide (or of the DNAencoding the same) are polypeptides which may be modified or altered inone or more amino acids (or in one or more nucleotides) such that thepeptide (or the nucleic acid) is not identical to the wild-typesequence, but has homology to the wild type polypeptide (or the nucleicacid).

A “mutation” of a polypeptide (or of the DNA encoding the same) is amodification or alteration of one or more amino acids (or in one or morenucleotides) such that the peptide (or nucleic acid) is not identical tothe sequences recited herein, but has homology to the wild typepolypeptide (or the nucleic acid).

As used herein, the term “imaging agent” can refer to a biological orchemical moiety capable of be encapsulated by a nanoparticle ormicroparticle construct of the application and that may be used todetect, image, and/or monitor the presence and/or progression of a cellcycle, cell function/physiology, condition, pathological disorder and/ordisease.

As used herein, the terms “treating” or “treatment” of a disease canrefer to executing a treatment protocol to increase blood flow by ameasurable amount from a thrombosed value. Thus, “treating” or“treatment” does not require complete restoration of blood flow from athrombosed value.

As used herein, the term “targeting moiety” can refer to a molecule ormolecules that are able to bind to and complex with a biomarker. Theterm can also refer to a functional group that serves to target ordirect a therapeutic agent to a particular location, cell type, diseasedtissue, or association. In general, a “targeting moiety” can be directedagainst a biomarker.

An “effective amount” can refer to that amount of a therapeutic agentthat results in amelioration of symptoms or a prolongation of survivalin the subject and relieves, to some extent, one or more symptoms of thedisease or returns to normal (either partially or completely) one ormore physiological or biochemical parameters associated with orcausative of the disease. “Therapeutic agents” can include any agent(e.g., molecule, drug, pharmaceutical composition, etc.) capable of beencapsulated by a nanoparticle or microparticle construct of theapplication and further capable of preventing, inhibiting, or arrestingthe symptoms and/or progression of a disease.

“Nanoparticle” or “microparticle” as used herein is meant to includeparticles, spheres, capsules, and other structures having a length ordiameter of about 10 nm to about 100 m. For the purposes of thisapplication, the terms “nanosphere”, “nanoparticle”, “nanoparticleconstruct”, “nanovehicle”, “nanocapsule”, “microsphere”,“microparticle”, and “microcapsule” are used interchangeably.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partialnumbers within that range, for example, 1, 2, 3, 4, 5, 5.5 and 6. Thisapplies regardless of the breadth of the range.

This application relates to nanoparticles or microparticles and theiruse in binding activated platelets with sufficient strength so that theyremain stably attached under the wall shear of hemodynamic flow as wellas to compositions and methods useful in the delivery of therapeuticand/or imaging agents to activated platelets. The nanoparticle ormicroparticle compositions described herein, integrate multiple-ligandadhesion-promoting functionality on a single nanoparticle ormicroparticle. It was found that heteromultivalently ligand-modifiednanoparticle or microparticle vehicles can simultaneously targetintegrins GPIIb-IIIa and P-selectin on thrombosis-associated activeplatelets, and can enhance selective binding to activated platelets at aclot site and stay retained even under hemodynamic flow.

It was further found that multi-receptor targeted strategy results inhigher binding and retention of nanoparticles or microparticles onactive platelets under flow conditions, compared to single receptortargeting at the same mol %. In addition, multi-receptor targeting by asingle nanoparticle or microparticle composition allows for enhancedbinding and retention with lower mol % of total ligands on the surfaceof a nanoparticle or microparticle, compared to single receptortargeting with only one type of ligand. Without being bound by theory itis believed that the enhanced effects of the heteromultivalentlyligand-modified nanoparticle or microparticle vehicles is due to avidityeffects of the dual receptor binding mechanisms. Therefore, the use ofthe heteromultivalent nanoparticle or microparticle compositionsdescribed herein can enhance the availability, safety and efficacy ofvascular disease therapy.

The compositions described herein can be used in diagnostic,therapeutic, and/or theranostic applications to deliver therapeuticagents and/or imaging agents to activated platelets and/or nearbytissues in a subject as well as selectively target activated plateletsand/or tissue of a subject upon systemic administration (e.g.,intravenous, intravascular, intraarterial infusion) of the compositionsto the subject. The compositions can also be remotely activated with aremote energy source to selectively release therapeutic agents and/orimaging agents to targeted activated platelets and/or nearby tissues ofthe subject.

In some embodiments, the compositions described herein can include abiocompatible, biodegradable nanoparticle or microparticle coreconstruct and a plurality of at least two types of unique targetingmoieties (e.g., a first and a second activated platelet targetingmoiety), each selective for separate activated platelet receptor exposedon the platelet surface. The plurality of targeting moieties are boundto, conjugated to, and/or decorated on the surface defined by thenanoparticle or microparticle core. The first and second activatedplatelet targeting moieties can be spatially or topographically arrangedon the nanoparticle or microparticle surface such that the first andsecond activated platelet targeting moieties do not spatially mask eachother and the nanoparticle or microparticle is able to bind to anactivated platelet with exposed activated platelet receptors, therebyenhancing retention of the nanoparticle or microparticle construct ontoactivated platelets under hemodynamic flow.

The nanoparticles or microparticles can be made from any biocompatible,biodegradable material that can form a nanoparticle or microparticle towhich the activated platelet targeting moieties described herein can beattached, conjugated, and/or decorated. Examples of nanoparticles ormicroparticles can include liposomes, lipidic nanoparticles, a hydrogel,micelle, metal nanoparticles, polymer nanoparticles, dendrimer, quantumdots, and/or combinations of these materials which can include and/or besurface modified or engineered with the activated platelet targetingmoieties. In some embodiments, the nanoparticles or microparticles canbe optically or magnetically detectable. In other embodiments,intrinsically fluorescent or luminescent nanoparticles ormicroparticles, nanoparticles or microparticles that comprisefluorescent or luminescent moieties, plasmon resonant nanoparticles, andmagnetic nanoparticles are among the detectable nanoparticles that canbe used.

The nanoparticles or microparticles can have a maximum length ordiameter of about 100 nm to about 10 m In general, the nanoparticle ormicroparticle construct can have dimensions small enough to allow thecomposition to be systemically administered to a subject and targeted toactivated platelets and tissue of the subject. In some embodiments, thenanoparticle or microparticle construct can have a size that facilitatesencapsulation of one or more therapeutic and/or imaging agents.

The nanoparticles or microparticles of the composition may be uniform(e.g., being about the same size) or of variable size. Particles may beany shape (e.g., spherical or rod shaped), but are preferably made ofregularly shaped material (e.g., spherical). Other geometries caninclude substantially spherical, circular, triangle, quasi-triangle,square, rectangular, hexagonal, oval, elliptical, rectangular withsemi-circles or triangles and the like. Selection of suitable materialsand geometries are known in the art.

In other embodiments, the nanoparticles or microparticles can includelipidic nanoparticles or microparticles, polymer nanoparticles ormicroparticles, liposomes, and/or dendrimers with a membrane, shell, orsurface that is formed from a naturally-occurring, synthetic orsemi-synthetic (i.e., modified natural) material. In some embodiments,the lipidic nanoparticles or liposomes can include a membrane or shellthat is formed from a naturally-occurring, synthetic or semi-syntheticmaterial that is generally amphipathic (i.e., including a hydrophiliccomponent and a hydrophobic component). Examples of materials that canbe used to form the membrane or shell of the lipidic nanoparticle ormicroparticle or liposome include lipids, such as fatty acids, neutralfats, phospholipids, oils, glycolipids, surfactants, aliphatic alcohols,waxes, terpenes and steroids. Semi-synthetic or modified natural lipidscan include natural lipids that have been chemically modified in somefashion. The lipid can be neutrally-charged, negatively-charged (i.e.,anionic), or positively-charged (i.e., cationic). Examples of anioniclipids can include phosphatidic acid, phosphatidyl glycerol, and fattyacid esters thereof, amides of phosphatidyl ethanolamine, such asanandamides and methanandamides, phosphatidyl serine, phosphatidylinositol and fatty acid esters thereof, cardiolipin, phosphatidylethylene glycol, acidic lysolipids, sulfolipids and sulfatides, freefatty acids, both saturated and unsaturated, and negatively-chargedderivatives thereof. Examples of cationic lipids can includeN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium chloride andcommon natural lipids derivatized to contain one or more basicfunctional groups.

Other examples of lipids, any one or combination of which may be used toform the membrane or shell of the lipidic nano-particle or liposome, caninclude: phosphocholines, such as 1-alkyl-2-acetoyl-sn-glycero3-phosphocholines, and 1-alkyl-2-hydroxy-sn-glycero 3-phosphocholines;phosphatidylcholine with both saturated and unsaturated lipids,including dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), and diarachidonylphosphatidylcholine (DAPC);phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine (DPPE), anddistearoylphosphatidylethanolamine (DSPE); phosphatidylserine;phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG);phosphatidylinositol; sphingolipids, such as sphingomyelin; glycolipids,such as ganglioside GM1 and GM2; glucolipids; sulfatides;glycosphingolipids; phosphatidic acids, such as dipalmitoylphosphatidicacid (DPPA) and distearoylphosphatidic acid (DSPA); palmitic acid;stearic acid; arachidonic acid; oleic acid; lipids bearing polymers,such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethyleneglycol (PEG); lipids bearing sulfonated mono-, di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate, and cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether andester-linked fatty acids; polymerized lipids (a wide variety of whichare well known in the art); diacetyl phosphate; dicetyl phosphate;stearylaamine; cardiolipin; phospholipids with short chain fatty acidsof about 6 to about 8 carbons in length; synthetic phospholipids withasymmetric acyl chains, such as, for example, one acyl chain of about 6carbons and another acyl chain of about 12 carbons; ceramides; non-ionicliposomes including niosomes, such as polyoxyalkylene (e.g.,polyoxyethylene) fatty acid esters, polyoxyalkylene (e.g.,polyoxyethylene) fatty alcohols, polyoxyalkylene (e.g., polyoxyethylene)fatty alcohol ethers, polyoxyalkylene (e.g., polyoxyethylene) sorbitanfatty acid esters (such as, for example, the class of compounds referredto as TWEEN (commercially available from ICI Americas, Inc., Wilmington,Del.), glycerol polyethylene glycol oxystearate, glycerol polyethyleneglycol ricinoleate, alkyloxylated (e.g., ethoxylated) soybean sterols,alkyloxylated (e.g., ethoxylated) castor oil,polyoxyethylene-polyoxypropylene polymers, and polyoxyalkylene (e.g.,polyoxyethylene) fatty acid stearates; sterol aliphatic acid estersincluding cholesterol sulfate, cholesterol butyrate, cholesterolisobutyrate, cholesterol palmitate, cholesterol stearate, lanosterolacetate, ergosterol palmitate, and phytosterol n-butyrate; sterol estersof sugar acids including cholesterol glucuronide, lanosterolglucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide,cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate;esters of sugar acids and alcohols including lauryl glucuronide,stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoylgluconate, and stearoyl gluconate; esters of sugars and aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid and polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, anddigitoxigenin; glycerol dilaurate, glycerol trilaurate, glyceroldipalmitate, glycerol and glycerol esters including glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate; long chain alcohols includingn-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, andn-octadecyl alcohol;6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmiticacid; cholesteryl(4′-trimethylammonio)butanoate;N-succinyldioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycerophosphoethanolamine andpalmitoylhomocysteine; and/or any combinations thereof.

Examples of biocompatible, biodegradable polymers that can be used toform the nanoparticles are poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s,poly(alkylene alkylate)s, copolymers of polyethylene glycol andpoly(lactide)s or poly(lactide-co-glycolide)s, biodegradablepolyurethanes, and blends and/or copolymers thereof.

Other examples of materials that may be used to form the nanoparticlesor microparticles can include chitosan, poly(ethylene oxide),poly(lactic acid), poly(acrylic acid), poly(vinyl alcohol),poly(urethane), poly(N-isopropyl acrylamide), poly(vinyl pyrrolidone)(PVP), poly(methacrylic acid), poly(p-styrene carboxylic acid),poly(p-styrenesulfonic acid), poly(vinylsulfonicacid),poly(ethyleneimine), poly(vinylamine), poly(anhydride), poly(L-lysine),poly(L-glutamic acid), poly(gamma-glutamic acid), poly(carprolactone),polylactide, poly(ethylene), poly(propylene), poly(glycolide),poly(lactide-co-glycolide), poly(amide), poly(hydroxylacid),poly(sulfone), poly(amine), poly(saccharide), poly(HEMA),poly(anhydride), gelatin, glycosaminoglycans (GAG), poly(hyaluronicacid), poly(sodium alginate), alginate, albumin, hyaluronan, agarose,polyhydroxybutyrate (PHB), copolymers thereof, and blends thereof.

In some embodiments, the nanoparticle or microparticle can include aliposome. The liposome can be an unilamellar liposome. The liposome canhave a width less than about 200 nm. For example, the width of theliposome can be about 100 nm to about 150 nm. In some embodiments, theliposome is about 150 nm in diameter. The liposome can have a highcholesterol content (e.g., about 40%) in the membrane in order toefficiently encapsulate a water-soluble thrombolytic drug protecting thedrug from plasma deactivation in circulation and prevent premature drugleakage due to membrane rigidity.

In an embodiment of the application, liposome nanoparticles having adiameter of 150 nm can be prepared by homogenizing DSPC (49 mol %), andDSPE-PEG-peptide (5 mol %), cholesterol (45 mol %) in 1:1chloroform:methanol and subjecting the mixture to reverse phaseevaporation through several cycles of freeze-thaw, followed by extrusionthrough a 200 nm polycarbonate membrane to achieve unilamellar vesicles.The particles can then be surface-modified with the targeting moietiesat a surface density and at a ratio of first activated targeting moietyto second activated targeting moiety effective to promote maximumparticle adhesion to exposed activated platelet surfaces and retentionat low-to-high sheer stresses.

In some embodiments, the nanoparticles can include quantum dots, i.e.,bright, fluorescent nanocrystals with physical dimensions small enoughsuch that the effect of quantum confinement gives rise to unique opticaland electronic properties. In certain embodiments, the nanoparticles areoptically detectable nanoparticles, such as metal nanoparticles. Metalsused to form the nanoparticles include, but not limited to, Ag, Au, Cu,Al, Fe, Co, Ni, Ru, Rh, Pd, and Pt or oxides thereof. In anotherembodiment, the metal comprises Fe or iron oxide. A further surfacefunctional layer can be added or formed in combination with a metal corematerial. Such functional layers can include, but are not limited to, Agoxide, Au oxide, SiO₂, Al₂O₃, Si₃N₄, Ta₂O, TiO₂, ZnO, ZrO₂, HfO₂, Y₂O₃,tin oxide, antimony oxide, iron oxide, and other oxides; Ag doped withchlorine or chloride, Au doped chlorine or chloride, Ethylene andChlorotrifluoroethylene (ECTFE), Poly(ethylene-co-butylacrylate-co-carbon monoxide) (PEBA), Poly(allylamine hydrochloride)(PAH), Polystyrene sulfonate (PSS), Polytetrafluoroethylene (PTFE),Polyvinyl alcohol (PVA), Polyvinyl chloride (PVC), Polyvinyldenefluoride (PVDF), Polyvinylprorolidone (PVP), and other polymers; stackedmultiple layers at least two layers including above listed metal layersand non-metal layers, and the like. In some embodiments, the metal corecan be Au, Ag, Fe, Ti, Ni, Cr, Pt, Ru, NiCr alloy, NiCrN, PtRh alloy,CuAuCo alloy, IrRh alloy and/or WRe alloy. The metals used should bebiocompatible.

In some embodiments, the nanoparticle or microparticle can be a magneticnanoparticle or microparticle. “Magnetic particles” refers tomagnetically responsive particles that contain one or more metals oroxides or hydroxides thereof. Compositions including opticallydetectable metal nano-particles or quantum dots can be detected in vivoupon systemic administration to a subject using magnetic resonanceimaging (MRI), magnetic resonance spectroscopy (MRS), nuclear magneticresonance imaging (NMR), multimodal imaging, fluorescent, positronemission tomography (PET), near infrared (NIR) imaging, X-ray imaging,and computed tomography (CT).

The composition of the application includes a first and a secondactivated platelet targeting moiety, each capable of targeting and/oradhering the nanoparticle or microparticle to an activated platelet viaa unique activated platelet surface biomarker. The targeting moietiescan comprise any molecule, or complex of molecules, which is/are capableof interacting with an exposed activated platelet surface biomarker. Thebiomarker can include, for example, a cellular protease, a kinase, aprotein, a cell surface receptor, a lipid, and/or fatty acid. Thetargeting moieties can interact with the biomarkers through, forexample, non-covalent binding, covalent binding, hydrogen binding, vander Waals forces, ionic bonds, hydrophobic interactions, electrostaticinteraction, and/or combinations thereof.

The targeting moieties can include, but are not limited to, syntheticcompounds, natural compounds or products, macromolecular entities,bioengineered molecules (e.g., polypeptides, lipids, polynucleotides,antibodies, antibody fragments), and small entities (e.g., smallmolecules, neurotransmitters, substrates, ligands, hormones andelemental compounds).

In some embodiments, the targeting moiety may comprise a peptide ligandmolecule, including, for example, ligands which naturally recognize aspecific desired receptor of an activated platelet, such as a fibrinogenligand specific for GPIIb-IIIa or a P-selectin glycoprotein ligand-1specific for p-selectin. Natural ligands binding to these two receptorsare responsible for stabilizing active platelet interactions at vasculardisease sites under a hemodynamic shear environment. Such ligandmolecules for use in a composition of the application include ligandsthat have been modified to increase their specificity of interactionwith a target receptor, ligands that have been modified to interact witha desired receptor not naturally recognized by the ligand, and fragmentsof such ligands. Peptide ligands can also include small protein-likechains designed to mimic a peptide ligand (peptidomimetics). In someembodiments, the targeting moiety can include a small molecule peptideligand. Preparation of peptide ligands can be accomplished by any numberof methods for generating peptides.

Advantageously, peptide targeting moieties can each include about 5 toabout 30 amino acids. By limiting the size of the peptides to about 5 toabout 30 amino acids, the first and second activated platelet targetingmoieties can be spatially or topographically arranged on thenanoparticle or microparticle surface such that the first and secondplatelet targeting moieties do not spatially mask each other and areable to adhere to an activated platelet surface with exposed surfacereceptors and enhance adhesion and retention of the nanoparticle ormicroparticle to the activated platelet surface at low-to-high sheerstresses.

In some embodiments, the first or second targeting moiety can be atargeting peptide comprising glycoprotein ligands having specificity toP-selectin. In some embodiments, the first or second targeting moietycan be a p-selectin targeting peptide comprising an amino acid sequenceof EWVDV (SEQ ID NO:1). In some embodiments, the first or secondtargeting moiety can be a p-selectin targeting peptide comprising anamino acid sequence of CDVEWVDVS (SEQ ID NO:4). In some embodiments, thefirst or second targeting moiety can be a p-selectin targeting peptidecomprising an amino acid sequence of DAEWVDVS (SEQ ID NO:5). The peptidemay be synthesized by any method known in the art. For example, thep-selectin targeting peptide may be synthesized using Fmoc based solidphase chemistry and characterized using MALDI-TOF mass spectroscopy.

In some embodiments, the first or second targeting moiety can be atargeting peptide comprising a tripeptide RGD (arginine-glycine-asparticacid) amino acid sequence motif having a high selective affinity toGPIIb-IIIa. GPIIb-IIIa is an integrin upregulated and stimulated into aligand-binding conformation on the surface of activated platelets. TheRGD motif containing targeting peptide may contain a single repeat ofthe RGD motif or may contain multiple repeats of the RGD motif, such as,for example, 2, or 5, or 10 or more repeats of the RGD motif. One ofskill in the art will understand that conservative substitutions ofparticular amino acid residues of the RGD motif containing targetingpeptide may be used so long as the RGD motif containing targetingpeptide retains the ability to bind comparably as the native RGD motif.One of skill in the art will also understand that conservativesubstitutions of particular amino acid residues flanking the RGD motifso long as the RGD motif containing targeting peptide retains theability to bind comparably as the native RGD motif. A RGD peptidecontaining targeting moiety can be synthesized using FMoc-based solidphase chemistry on resin, and characterized using mass spectroscopy.

An RGD peptide having high selective affinity to GPIIb-IIIa can includea linear-RGD peptide or a cyclic RGD peptide. Preferred RGD peptides donot bind or activate quiescent platelets nor interact with otherRGD-binding integrins. In some embodiments, the targeting moiety caninclude a linear RGD (lRGD) peptide having the amino acid sequence of(SEQ ID NO:2). In some embodiments, a first of second targeting moietycan include a cyclic RGD (abbreviated cRGD) peptide having the aminoacid sequence of cyclo-CNPRGDY(-OEt)RC (SEQ ID NO: 3). For cRGDsynthesis, the terminal cysteine residues of the linear precursor can becyclized by a disulfide bond using an ferricyanide-mediated oxidationprocess.

The targeting moiety can be coupled to nanoparticles or microparticlesof the composition using a linker. The linker can be of any suitablelength and contain any suitable number of atoms and/or subunits. Thelinker can include one or a combination of chemical and/or biologicalmoieties. Examples of chemical moieties can include alkyl groups,methylene carbon chains, ether, polyether, alkyl amide linkers, alkenylchains, alkynyl chains, disulfide groups, and polymers, such aspoly(ethylene glycol) (PEG), functionalized PEG, PEG-chelant polymers,dendritic polymers, and combinations thereof. Examples of biologicalmoieties can include peptides, modified peptides, streptavidin-biotin oravidin-biotin, polyaminoacids (e.g., polylysine), polysaccharides,glycosaminoglycans, oligonucleotides, phospholipid derivatives, andcombinations thereof.

The first and second activated platelet targeting moieties can betethered to the surface of the nanoparticle or microparticle construct.In some embodiments, the first and second activated platelet targetingmoieties can be tethered to the surface of the nanoparticle ormicroparticle construct via a polymer tether. The polymer tether can belinked to the nanoparticle or microparticle construct directly orindirectly by any means. For example, the polymer tether can be linkedto the nanoparticle or microparticle construct using a covalent link, anon-covalent link, an ionic link, and a chelated link, as well as beingabsorbed or adsorbed onto the nanoparticle or microparticle construct.In addition, the polymer tether can be linked to the nanoparticle ormicroparticle construct through hydrophobic interactions, hydrophilicinteractions, charge-charge interactions, r-stacking interactions,combinations thereof, and like interactions.

In some embodiments, the targeting moieties are conjugated to thesurface of the nanoparticle or microparticle via a PEG molecule linker.The PEG molecules can have a variety of lengths and molecular weights,including, for example, PEG 200, PEG 1000, PEG 1500, PEG 4600, PEG10,000, or combinations thereof. In other embodiments, the targetingmoieties can be conjugated to the nanoparticle or microparticle surfacewith PEG acrylate, or PEG diacrylate, molecules of a variety ofmolecular weights. Active platelet-targeting moieties can be conjugatedon the liposome surface, to promote specific binding and retention ofthe liposomes on thrombus-associated active platelets. For example,integrin GPIIb-IIIa and P-selectin targeting small molecular weightpeptide targeting moieties can be conjugated on the surface of aliposome via a PEG spacer.

Targeting peptides can be conjugated ‘on resin’ to NHS-estermodified-lipid (DSPE-PEG-NHS ester) by reductive amidation to formDSPE-PEG-peptide (see FIG. 2). The PEG-peptide conjugates or PEGylatedpeptides can then be conjugated to the nanoparticle or microparticleusing known conjugation techniques.

The ratio of the first targeting moiety to the second targeting moietyprovided on the nanoparticle or microparticle surface can be about 80:20to about 20:80 and be adjusted accordingly to maximize adhesion underlow-to-high shear conditions. It will be appreciated, that other ratioscan be used to enhance the nanoparticle or microparticle adherence andretention to activated platelets. In addition, the total mol % oftargeting moieties used for particle surface-modification can be variedto enhance binding and retention to activated platelets under flow.

In other embodiments, the composition can further include imaging agents(or detectable moieties) and/or therapeutic agents that are encapsulatedby (e.g., within liposome, lipidic nanoparticle or microparticle, orpolymer nanoparticle or microparticle), contained in (e.g., polymernanoparticles or dendrimers), or conjugated to the nanoparticles.

Imaging agents can include any substance that may be used for imaging ordetecting a region of interest (ROI) in a subject and/or diagnosing thepresence or absence of a disease or diseased tissue in a subject. Theimaging agent can be selected such that it generates a signal, which canbe measured and whose intensity is related (preferably proportional) tothe distribution of the imaging agent and activated platelets in thesubject. Examples of imaging agents include, but are not limited to:radionuclides, fluorescent dyes, chemiluminescent agents, colorimetriclabels, and magnetic labels. In one example, the imaging agent caninclude a radiolabel that is detected using gamma imaging whereinemitted gamma irradiation of the appropriate wavelength is detected.Methods of gamma imaging include, but are not limited to, SPECT and PET.For SPECT detection, the chosen radiolabel can lack a particularemission, but will produce a large number of photons in, for example, a140-200 keV range. For PET detection, the radiolabel can be apositron-emitting moiety, such as 19F.

In another example, the imaging agent can include an MRS/MRI radiolabel,such as gadolinium, 19F, 13C, that is coupled (e.g., attached orcomplexed) with the composition using general organic chemistrytechniques. The imaging agent can also include radiolabels, such as 18F,11C, 75Br, or 76Br for PET by techniques well known in the art and aredescribed by Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY ANDAUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds.)391-450 (Raven Press, N Y 1986) the contents of which are herebyincorporated by reference. The imaging can also include 1231 for SPECT.

The imaging agent can further include known metal radiolabels, such asTechnetium-99m (99mTc). Preparing radiolabeled derivatives of Tc99m iswell known in the art. See, for example, Zhuang et al., “Neutral andstereospecific Tc-99m complexes:[99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)”Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small andneutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developingnew brain imaging agents” Nuclear Medicine & Biology 25(2):135-40,(1998); and Hom et al., “Technetium-99m-labeled receptor-specificsmall-molecule radiopharmaceuticals: recent developments and encouragingresults” Nuclear Medicine & Biology 24(6):485-98, (1997).

Therapeutic agents or bioactive agents, encapsulated by, contained in,and/or linked to nanoparticles or microparticles of the presentapplication can include any substance capable of exerting a biologicalor therapeutic effect in vitro and/or in vivo. Therapeutic agents canalso include any therapeutic or prophylactic agent used in the treatment(including the prevention, diagnosis, alleviation, or cure) of a malady,affliction, condition, disease or injury in a subject. Examples oftherapeutic agents include, but are not limited to thrombolytic,anti-thrombosis, and anti-proliferative agents. The therapeutic agentscan be in the form of biologically active ligands, small molecules,peptides, polypeptides, proteins, DNA fragments, DNA plasmids,interfering RNA molecules, such as siRNAs, oligonucleotides, and DNAencoding for shRNA.

In some embodiments, the therapeutic agent can be a thrombolytic agentthat is encapsulated by, contained in, and/or linked to thenanoparticles or microparticles. Thrombolytic agents are used todissolve blood clots in a procedure termed thrombolysis and can limitthe damage caused by the blockage or occlusion of a blood vessel.Thrombolytic agents can include analogs of tissue plasminogen activator(tPA), the protein that normally activates plasmin and recombinanttissue plasminogen activators (r-tPAs) include alteplase, reteplase, andtenecteplase (sold under the trade name TNKase) and desmoteplase.Additional thrombolytic agents include anistreplase (sold under thetrade name EMINASE), streptokinase (sold under the trade namesKABIKINASE, STREPTASE), and urokinase (sold under the trade nameABBOKINASE).

In some embodiments, the therapeutic agent can be an anti-thromboticagent that is encapsulated by, contained in, and/or linked to thenanoparticles or microparticles. Antithrombotic agents can includeanticoagulants and antiplatelet agents.

Anticoagulants slow down clotting, thereby reducing fibrin formation andpreventing clots from forming and growing. Anticoagulants includecoumarins (vitamin K antagonists) such as coumadin. Anticoagulants alsoinclude but are not limited to heparin, heparin derivatives and directthrombin inhibitors including the bivalent drugs hirudin, lepirudin, andbivalirudin and the monovalent drugs argatroban and dabigatran.

Antiplatelet agents prevent platelets from clumping and also preventclots from forming and growing. Antiplatelet agents can include but arenot limited to aspirin and clopidogrel (sold under the trade namePLAVIX).

In some embodiments, the therapeutic and/or imaging agents can be loadedinto and/or onto the nanoparticles or microparticles by encapsulation,absorption, adsorption, and/or non-covalent linkage of the agent to orwithin the nanoparticle or microparticle. The amount of agent loadedonto or in the nanoparticle or microparticle can be controlled bychanging the size of the nanoparticle or microparticle or thecomposition of the nanoparticle or microparticle.

In some embodiments, release of the therapeutic or imaging agent fromthe nanoparticle or microparticle of the composition can occur bydesorption, diffusion through the polymer or lipid coating, or polymeror lipid wall, nanoparticle or microparticle erosion, and/or disruptionof the nanoparticle or microparticle structure, which can all becontrolled by the type of the nanoparticle or microparticle, i.e.,having it become swollen or degradable in the chosen microenvironment.

In some embodiments, the therapeutic or imaging agent can be releasedfrom the nanoparticle or microparticle composition through the use of aninternal and/or external trigger. Internal triggers include the body'sinternal pH, chemical and enzymatic activity. External triggers caninclude light and ultrasound.

Advantageously, a nanoparticle or microparticle construct that allowsremote release of the therapeutic agent, (e.g., a thrombolytic agent,such as tPA) can target or be targeted to activated platelets of athrombus associated with a vascular disease site, by systemicadministration (e.g., intravenous, intravascular, or intraarterialinfusion) to the subject and once targeted to the site remotely releasedto specifically treat the targeted activated platelets or disease sitetissue of the subject (e.g., activated platelets of a thrombusassociated with a vascular disease site). Targeting and selectiverelease of the thrombolytic agents and/or anti-thrombotic agents toactivated platelets and the surrounding cells and tissue allowstreatment of such vascular disease using thrombolytic agents and/oranti-thrombotic agents, which would provide an otherwise diminishedtherapeutic effect if not targeted and remotely released using thecompositions described herein.

In some embodiments, release of the therapeutic agent and/or imagingagent from the nanoparticle or microparticle of the composition can betriggered by an energy source that supplies energy to the compositioneffective to release the therapeutic agent or imaging agent from thenanoparticle or microparticle construct. The energy source can beexternal or remote from a subject, which allows non-invasive remoterelease of the therapeutic agent to the subject. The remote energysource can be, for example, a minimally invasive laser that can beinserted in vivo in the subject being treated or positioned external orex vivo the subject. The energy from laser can be in the near infraredrange to allow deep radiation penetration into tissue and remote releaseof therapeutic agent or imaging agent.

Therefore, in some embodiments, a nanoparticle or microparticleconstruct of the composition can be surface modified to be responsive toenergy, from a remote source that is effective to release thetherapeutic agent from the nanoparticle or microparticle upon mechanicaldisruption of the nanoparticle or microparticle membrane or shell afteradministering the composition to a subject.

In an exemplary embodiment, near infra-red (NIR)-responsive goldnanorods (GNRs) conjugated close to the surface of the heteromultivalentnanoparticles encapsulating or containing a therapeutic agent canexhibit plasmon resonance phenomena under tissue-penetrating NIR light,such that the resultant thermo-mechanical energy dissipation results indisruption of the nanoparticle or microparticle to render site-selectiverapid drug release. Thus, in some embodiments, NIR-irradiation fromspecialized external or catheter-mediated laser devices can be used toremotely trigger rapid drug release at the targeted disease site viaphotothermal destabilization of GNR-modified nanoparticles.

Upon administration of the composition to a subject by, for example,intravascular administration, the composition can target an activatedplatelet at a vascular disease site being treated. In some embodiments,the composition can be imaged by, for example, magnetic resonanceimaging or computed tomography, to confirm localization and targeting ofthe composition to the occlusive vascular disease site. The compositiontargeted to the vascular disease site can be applied NIR from a remoteNIR energy source that is external to the subject being treated tomechanically resonate or oscillate the GNRs on the nanoparticle ormicroparticle and rapidly release the therapeutic agent from theliposome membrane or shell due to defects in the membrane or shellcaused by oscillation of the gold linked to the nanoparticle ormicroparticle.

GNRs of certain dimensions exhibit plasmon resonance in the NIRwavelength range (700-900 nm), resulting in generation of heat. GNRs ofa NIR-responsive aspect ratio can be synthesized relatively consistentlyvia kinetic controls. NIR-responsive GNR can be fabricated as shown inthe Examples below using a seed-mediated method. ExemplaryNIR-responsive GNRs are about 50 nm long with an aspect ratio of about2-5 that have plasmon resonance in the NIR range.

In some embodiments, the mild NIR energy applied to the nanoparticle ormicroparticle of the composition can be that amount effective cause theGNRs on the nanoparticles to mechanically resonate or oscillate at anamount or level effective to disrupt the nanoparticle or microparticlemembrane or shell and release the therapeutic agent without causingsignificant heating (e.g., greater than 1° C., 2° C., 3° C., or 5° C.)around the nanoparticle or microparticle when administered to a subject.

In an exemplary embodiment, GNR-decorated ligand modifiedplatelet-targeted thrombolytic-loaded liposomes can be exposed to a 800nm laser diode driven at ˜1W power and at frequencies of 0.5, 5, 20,100, 200 and 500 kHZ to provide pulse widths of 200, 20, 5, 1, 0.5 and0.2 μs. The NIR irradiation exposure durations can be maintained atabout 10 sec to about 10 min for the various pulse widths. In someembodiments the NIR irradiation exposure durations can be maintained atabout 30 sec to about 5 min.

NIR-responsive GNRs can be fabricated and conjugated close to thenanoparticle or microparticle (e.g., liposome) surface using lipoic acidlinkers, to allow NIR-induced photothermal disruption of thenanoparticle or microparticle for payload release. In some embodiments,the nanoparticles will have the GNRs conjugated close to their surfacevia the lipoic acid linkers and the platelet-targeting peptidesconjugated above the surface via PEG linkers.

GNR-modified liposomes can be produced, for example, by formulatingtogether DSPE-lipoic acid, DSPE-PEG-cRGD and DSPE-PEG-EWVDV along with40 mol % cholesterol and remaining mol % of DSPC. The liposome surfacelipoic acid motifs can then be treated with DTT to generate thiols andthen decorated with the GNRs (see FIG. 13). GNR decoration density canbe optimized for favorable NIR-induced payload release kinetics.

Preferably, the GNR surface decoration parameters (corresponding toDSPE-lipoic acid mole %) on liposomes and the NIR-irradiation parametersare the parameters that result in at least 80% release of encapsulatedpayload upon NIR-exposure for short durations. The power, pulse widthsand frequencies are those safe for the intended in vivo applications.

It will be appreciated that other remote energy sources can be used torelease the therapeutic agent or imaging agent from the nanoparticle ormicroparticle and that the selection of the energy source will depend atleast in part on the nanoparticle or microparticle construct used toform the composition.

In some embodiments, the compositions comprising a multi-ligand modifiednanoparticle or microparticle described herein, can be formulated in apharmaceutical composition and administered to an animal, preferably ahuman, to facilitate the delivery of a therapeutic agent.

Formulation of pharmaceutical composition for use in the modes ofadministration noted below (and others) are described, for example, inRemington's Pharmaceutical Sciences (18^(th) edition), ed. A. Gennaro,1990, Mack Publishing Company, Easton, Pa.

Such a pharmaceutical composition may consist of a plurality of surfacemodified heteromultivalent nanoparticle or microparticle alone, in aform suitable for administration to a subject, or the pharmaceuticalcomposition may comprise a plurality of nanoparticles and one or morepharmaceutically acceptable carriers, one or more additionalingredients, one or more pharmaceutically acceptable therapeutic agents,bioactive agents, imaging/diagnostic agents, or some combination ofthese. In some embodiments, the therapeutic agent may be present in thepharmaceutical composition in the form of a physiologically acceptableester or salt, such as in combination with a physiologically acceptablecation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the therapeutic agent may be combinedand which, following the combination, can be used to administer thetherapeutic agent to a subject. As used herein, the term“physiologically acceptable” ester or salt means an ester or salt formof the therapeutic agent which is compatible with any other ingredientsof the pharmaceutical composition, which is not deleterious to thesubject to which the composition is to be administered.

For example, pharmaceutical compositions can be in the form of a sterileaqueous or oily injectable solution containing, if desired, additionalingredients, for example, preservatives, buffers, tonicity agents,antioxidants, stabilizers, nonionic wetting or clarifying agents, andviscosity increasing agents. This suspension or solution may beformulated according to the known art, and may comprise, in addition tothe therapeutic agent, additional ingredients such as the dispersingagents, wetting agents, or suspending agents described herein. Suchsterile injectable formulations may be prepared using a non-toxicparenterally acceptable diluent or solvent, such as water or 1,3 butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono or di-glycerides.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials.

In some embodiments, a bioactive agent, imaging/diagnostic agent, and/ortherapeutic agent can be conjugated, encapsulated, and/or contained withthe heteromultivalent nanoparticle or microparticle so that theheteromultivalent nanoparticle or microparticle acts as a deliveryvehicle. In other embodiments, the bioactive agent, imaging/diagnosticagent, and/or therapeutic agent can be merely contained in apharmaceutical composition either with or without the modifiednanoparticles and administered to concurrently with or separately fromadministration of the nanoparticles. Selection of a bioactive agent,imaging/diagnostic agent, and/or therapeutic agent to be conjugated toor encapsulated within the heteromultivalent nanoparticle ormicroparticle is dependent upon the use of the nanoparticle ormicroparticle and/or the condition being treated and the site and routeof administration.

In some embodiments, a composition described herein can be used in amethod for treating a vascular disease in a subject. In someembodiments, the disease can be characterized, in part, by the presenceof activated platelets and/or platelet aggregation at a disease site(e.g., a thrombosed site). In some embodiments, a therapeuticallyeffective amount of the composition can be administered in vivo to asubject to treat the subject. In some embodiments, an effective amountof the composition is the amount required to restore blood flow by about50% from its thrombosed value. Post-irradiated blood flow can bemonitored for example sonographically.

It should be understood, that the methods of treatment by the deliveryof a composition including a heteromultivalent nanoparticle ormicroparticle includes the treatment of subjects that are alreadyafflicted with a vascular disease or symptoms thereof (e.g., a bloodclot), as well as prophylactic treatment uses in subjects not yetafflicted and/or experiencing symptoms. In a preferred embodiment thesubject is an animal. In a more preferred embodiment the subject is ahuman.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally foradministration to animals of all sorts. Modification of pharmaceuticalcompositions for administration to humans in order to render thecompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, animals including commerciallyrelevant animals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

Pharmaceutical compositions that are useful in the methods describedherein may be administered by any convenient route, such as by infusionor bolus injection. For example, the composition may be introduced intothe subject by any suitable route, including intraventricular injectionor intraventricular injection via an intraventricular catheter that isattached to a reservoir.

The composition can be delivered systematically (e.g., intravenously),regionally, or locally by, for example, intraarterial, intrathrombal,intravenous, parenteral, intraneural cavity, topical, oral or localadministration, as well as subcutaneous, intra-tracheal (e.g., byaerosol), or transmucosal (e.g., buccal, bladder, vaginal, uterine,rectal, nasal, mucosal). If delivery of the composition to the brain isdesired, the targeted composition can be injected into an artery of thecarotid system of arteries (e.g., occipital artery, auricular artery,temporal artery, cerebral artery, maxillary artery etc.). As discussedabove, the composition can be formulated as a pharmaceutical compositionfor in vivo administration.

The pharmaceutical compositions described herein may also be formulatedso as to provide slow, prolonged or controlled release. In general, acontrolled-release preparation is a pharmaceutical composition capableof releasing the heteromultivalent nanoparticles at a desired orrequired rate to maintain constant activity for a desired or requiredperiod of time.

The relative amounts of the ingredients in a pharmaceutical compositionof the invention will vary, depending upon the identity, size, andcondition of the subject treated and further depending upon the route bywhich the composition is to be administered. By way of a non-limitingexample, the composition may comprise between 0.1% and 100% (w/w) of theheteromultivalent nanoparticles.

The composition including heteromultivalent nanoparticles can beadministered to the subject at an amount effective to provide a desiredresult(s) and to avoid undesirable physiological results. In someembodiments, the synthetic platelet compositions described herein may beadministered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.In one embodiment, a dose can be administered that results in aconcentration of the synthetic platelets between 1 μM and 1 μM in amammal. The precise dose to be employed can also depend on the route ofadministration, and should be decided according to the judgment of amedical practitioner and each subject's circumstances. In addition,known in vitro and in vivo assays may optionally be employed to helpidentify optimal dosage ranges. Effective doses may be extrapolated fromdose-response curves derived from in vitro or in vivo test systems.Preferably, the dosage of the heteromultivalent nanoparticles will varyfrom about 1 μg to about 50 mg per kilogram of body weight of theanimal. More preferably, the dosage will vary from about 10 μg to about15 mg per kilogram of body weight of the animal. Even more preferably,the dosage will vary from about 100 μg to about 10 mg per kilogram ofweight of the animal.

The composition can be administered in a variety of unit dosage forms,depending upon the particular disease or disorder being treated, thegeneral medical condition of each subject, the method of administration,and the like. Details on dosages are well described in the scientificliterature. The exact amount and concentration of the targetedcompositions, or the “effective dose”, can be routinely determined(e.g., by a medical practitioner).

The pharmaceutical composition may be administered to a subject asneeded. The pharmaceutical composition may be administered to a subjectas frequently as several times daily, or it may be administered lessfrequently, such as once a day, once a week, once every two weeks, oncea month, or even less frequently, such as once every several months oreven once a year or less. The “dosing regimen” will depend upon avariety of factors, such as the type and severity of the disease beingtreated, the general state of the subject's health, the subject's age,and the like. Using guidelines describing alternative dosing regimens,e.g., from the use of other agents and compositions, the skilled artisancan readily determine by routine trials the optimal effectiveconcentrations of the composition.

In some embodiments, the composition described herein can be used within vivo imaging methods where detection and imaging of activatedplatelets or an associated thrombus cannot readily be performed withtraditional optical detection or imaging techniques. These methods caninclude, for example, endovascular detection. It will be appreciatedthat the compositions can be used in other in vivo methods as well asintraoperative procedures.

For imaging methods, a plurality of the heteromultivalent nanoparticlescan be delivered to the targeted activated platelets and nearby tissueof the subject in vivo by administering an effective amount orconcentration of the compositions to the subject. By effective amount orconcentration of the composition, it is meant an amount of thecompositions that are effective for detecting and imaging the targetedactivated platelets or nearby tissue. As apparent to one skilled in theart, such an amount will vary depending on factors that include: theamount of tissue to be imaged; the rate of contact of the compositionswith the platelets; and any abnormalities of the platelets or thrombussite that may affect the efficiency of the nanoparticle or microparticleconstruct of the composition contacting or binding to the targetedactivated platelets.

In some embodiments, the heteromultivalent nanoparticle or microparticlecomposition can be administered to the subject by venous (or arterial)infusion. In venous infusion, an effective amount or concentration ofthe composition administered to subject can be that amount orconcentration that is detectable in the targeted platelets or associateddisease site after sequestration of the composition in the liver,spleen, and lymph nodes. Optionally, the composition can be administeredto the subject by directly injecting the nanoparticle or microparticleconstruct into the vasculature of the area being identified or an areaproximate or peripheral to the area being identified. Direct injectionof the nanoparticle or microparticle construct can be performed byusing, for example, a syringe.

In other embodiments, the nanoparticles can be administered to a subjectfor imaging at least one region of interest (ROI) of the subject. TheROI can include a particular area or portion of the subject and, in someinstances, two or more areas or portions throughout the entire subject.The ROI can include, for example, pulmonary regions, gastrointestinalregions, cardiovascular regions (including myocardial tissue), renalregions, as well as other bodily regions, tissues, lymphocytes,receptors, organs and the like, including the vasculature andcirculatory system, and as well as diseased tissue, including neoplasticor cancerous tissue. The ROI can include regions to be imaged for bothdiagnostic and therapeutic purposes. The ROI is typically internal;however, it will be appreciated that the ROI may additionally oralternatively be external. For example, the ROI can include a thrombussite or any occlusive vascular disease site in a subject.

At least one image of the ROI can be generated using an imaging modalityonce the targeted nanoparticles localize to the RO. The imaging modalitycan include one or a combination of known imaging techniques capable ofvisualizing the nanoparticles. Examples of imaging modalities caninclude ultrasound (US), magnetic resonance imaging (MRI), nuclearmagnetic resonance (NMR), computed topography (CT), electron spinresonance (ESR), nuclear medical imaging, optical imaging, and positronemission topography (PET).

In one example, the nanoparticles can be detected with MRI and/or x-ray.MRI relies upon changes in magnetic dipoles to perform detailed anatomicimaging and functional studies. For example, the electron dense core ofGNRs of the nanoparticle, can make them highly visible on X-ray,monochromatic X-ray, computed tomography (CT) and ultrasound (US).

Optionally, the nanoparticles can be further modified to facilitatedetection and imaging with MRI and CT as well as positron emissiontomography (PET). For MRI applications, gadolinium tags can be attachedto the shell. For PET applications, radioactive tags can be attached tonanoparticles. For CT applications, iodide or other heavy metals can beattached to the nanoparticles to facilitate CT contrast.

It will be appreciated the nanoparticles will likely be most usefulclinically when several imaging techniques or imaging followed by amedical or surgical procedure is used. In this way, the ability to useone agent for multiple imaging modalities is optimized making thenanoparticles cost-competitive with existing contrast agents.

For multimodal imaging applications, the nanoparticles can beadministered to a subject and then preoperatively imaged using, forexample, CT or MRI. After preoperative imaging, the nanoparticles canserve as optical beacons for use during surgery leading to more completeresections or more accurate biopsies. In surgical resection of lesions,the completeness of resection can be assessed with intra-operativeultrasound, CT, or MRI. For example, in glioma (brain tumor) surgery,the nanoparticles can be given intravenously about 24 hours prior topre-surgical stereotactic localization MRI. The nanoparticles can beimaged on gradient echo MRI sequences as a contrast agent that localizeswith a thrombus.

In many occlusive vascular diseases like stroke, myocardial infarction,peripheral arterial diseases and deep vein thrombosis, thrombo-occlusionand ischemia can deprive vital organs of circulation, oxygen andnutrients, leading to tissue and organ morbidity and often mortality.Rapid clot dissolution and revascularization is a mainstay in theclinical treatment of these critical disease conditions. Therefore, theapplication further relates to a method of treating a vascular diseasein a subject. Vascular diseases and injuries treatable by thenanoparticles described herein can include disease states or injuriescharacterized in part by the aggregation and/or adhesion of activatedplatelets in a disease site of subject. In some embodiments, thevascular disease or vascular injury includes an occlusive vasculardisease such as but not limited to stroke, myocardial infarction,peripheral arterial diseases and deep vein thrombosis, thrombo-occlusionand ischemia.

A composition comprising the nanoparticles described herein thatincludes a therapeutic agent for the treatment of vascular disease canbe formulated for administration (e.g., injection) to a subjectdiagnosed with at least one occlusive vascular disease. Thenanoparticles can be formulated according to a method as described aboveand include, for example, at least one therapeutic agent or imagingagent as well as a first and a second targeting moiety to target theactivated platelets at the occlusive vascular disease site. Theplatelet-targeted nanoparticles loaded with a therapeutic agent arefurther GNR-modified. It is contemplated that compositions of thepresent application can be administered via I.V.-injection and can stayin circulation long enough to recognize and bind at vascular sitesexhibiting platelet hyperactivity. Alternatively, the GNR-modifiedactivated platelet-targeted nanoparticle loaded with a therapeutic agentcan be administered via direct I.V. bolus injection at or near thedisease site of interest. After an amount of time necessary for thevehicle to bind to thrombotic activated platelets, therapeutic agentsencapsulated by the nanoparticles can be rapidly released remotely atthe targeted site by utilizing near-infrared (NIR-induced) photothermaldestabilization of the GNR-modified nanoparticles. Activeplatelet-targeted GNR-modified nanoparticles can undergothrombus-selective binding and retention under dynamic flow and allowNIR-triggered release of drugs for targeted thrombolytic therapy.

In some embodiments, compositions of the application are administeredimmediately after it has been determined they are clinicallyappropriate. The advantage of administration is highest within the firstsixty minutes after a thrombotic event, but may extend up to six hoursafter the start of symptoms. In some embodiments, compositions areadministered in combination with anticoagulant drugs such as intravenousheparin or low molecular weight heparin, for synergistic antithromboticeffects and secondary prevention.

The following examples are for the purpose of illustration only and arenot intended to limit the scope of the claims, which are appendedhereto.

Example 1

We have created nanoparticles platforms that bear two types of ligands(a GPIIb-IIIa-targeting peptide and a P-selectin-targeting peptide)together, for simultaneous targeting of GPIIb-IIIa and P-selectin. Wehave data to demonstrate that such combination targeting results inhighly enhanced platelet-selective shear-stable binding under flow,compared to any one type of targeting by itself. Such synergistictargeting mechanisms enable strong site-selective binding under flowingblood.

Natural ligands binding to these two receptors are responsible forstabilizing active platelet interactions at vascular disease sites undera hemodynamic shear environment. Integrin αIIbβ3 on activated platelets,in its stimulated ligand binding conformation, binds to the bi-dentateligand fibrinogen (Fg) to promote interlinking of active platelets inprimary thrombus formation. This integrin also allows interaction ofactivated platelets with diseased endothelial cells (ECs) viaFg-mediated crosstalk with integrin α_(v)β₃. P-selectins, expressedspecifically on activated platelets and stimulated endothelial cells viamembrane fusion of cytoplasmic granules, are responsible for allowinginteraction of platelets and ECs with monocytes via binding toP-selectin Glycoprotein Ligand-1 (PSGL-1) present on the surface ofmonocytes. P selectin also mediates interplatelet interactions viasulfatides and platelet interaction with endothelial cells via GlyCAM-1,CD34, and MadCAM 1, providing supplementary mechanisms ofplatelet-monocyte-EC interaction stabilization at vascular diseasesites. Hence, we rationalized that exploiting simultaneous binding tothese two receptors by our liposomal nanoconstructs will not onlyenhance vascular disease site-selectivity, but also provide sufficientbinding stability under hemodynamic flow at the target site.

For specific targeting to activated platelets, we have chosen integrinαIIbβ and P-selectin as our molecular target epitopes since they arepresent at high levels, specifically on the surface of activatedplatelets. This will enable high selectivity of our liposomalnanoconstructs toward activated platelets and not quiescent plateletsand, therefore, will enhance selectivity to vascular disease sites. Wehave previously demonstrated enhanced targeting of αIIbβ on activatedplatelets by decorating liposome surface with fibrinogen-mimetic RGDpeptides. In current research, we combined αIIbβ3-targeting by aGSSSGRGDSPA (SEQ ID NO:6) peptide with P selectin targeting by aDAEWVDVS (SEQ ID NO:5) peptide. We carried out heteromultivalentsurface-modification of the liposome membrane with these peptides andstudied the interaction (specific binding and retention) of theresultant nanoconstructs with collagen-adhered activated platelets in aparallel plate flow chamber (PPFC) at various wall shear values. Thebinding and retention of dual-targeted constructs were compared tonontargeted or singly-targeted constructs.

Materials and Methods Materials

Phosphate Buffered Saline (PBS), 3.8% w/v sodium citrate,parformaldehyde (PFA), Bovine Serum Albumin (BSA), Trifluoroacetic acid(TFA), chloroform, methanol, and ethanol were obtained from ThermoFisher Scientific (Pittsburgh, Pa., USA). Cholesterol, Ninhydrin,Phenol, Potassium cyanide, Pyridine, 1,2-Ethanedithiol (EDT),Thiolanisole, and collagen were obtained from Sigma Aldrich (St. Louis,Mo., USA). All amino acids were purchased from Advanced ChemTech(Louisville, Ky., USA). Polycarbonate filters with 200 nm pores wereobtained from Whatman (Kent, UK). Fluorescently-labeled monoclonalantibodies, namely, FITC-anti-CD41a (staining platelet GPIIb-IIIa) andAlexaFluor 647-anti-CD62P (staining activated platelet P-selectin), wereobtained from BD Biosciences (San Jose, Calif., USA) and BioLegend (SanDiego, Calif., USA), respectively. Adenosine Diphosphate (ADP) waspurchased from Bio/Data Corporation (Horsham, Pa., USA). The lipidsDistearyl Phosphatidyl Choline (DSPC), Distearyl PhosphatidylEthanolamine (DSPE), Polyethylene Glycolmodified DSPE (DSPE-PEG2000),DSPE-NHS ester, and Carboxy-Polyethylene Glycol-modified DSPE(DSPE-PEG2000-NHS ester) were obtained from Avanti Polar Lipids(Alabaster, Ala., USA). N-Hydroxysuccinimide (NHS)-modifiedfluorophores, namely, NHS-Fluorescein and NHS-Rhodamine were obtainedfrom Invitrogen Corporation (Carlsbad, Calif., USA). The Parallel PlateFlow Chamber (PPFC) system was purchased from Glycotech (Gaithersburg,Md., USA).

Peptide Synthesis and Fabrication of Peptide-Modified LiposomalNanoconstructs

An 11-residue linear RGD peptide, GSSSGRGDSPA (SEQ ID NO: 2), and an8-residue P-selectin targeting peptide, DAEWVDVS (SEQ ID NO:5), weresynthesized using Fmoc based solid phase chemistry and characterizedusing MALDI-TOF mass spectroscopy. Negative control peptide sequences,namely GSSSGRGESPA (SEQ ID NO:7) and DAEWVEVS (SEQ ID NO:8), weresynthesized and characterized in the same manner. All peptides wereconjugated ‘on resin’ to NHS-ester modified-lipid (DSPE-PEG-NHS ester)by reductive amidation to form DSPE-PEG-peptide (schematic in FIG. 2).Conjugation yield was determined to be 95.8±4.9% using a Ninhydrinassay. To synthesize DSPE-Fluorescein (green fluorescence, λmax˜530 nm)or DSPE-Rhodamine (red fluorescence, λmax ˜570 nm), the free NH₂ terminiof DSPE was reacted to the amine reactive NHS-Fluorescein orNHS-Rhodamine at a basic pH. The liposomes were fabricated byhomogenizing DSPC (49 mol %), DSPE-PEG-peptide (5 mol %), cholesterol(45 mol %) and DSPE-PEG-Rhodamine or Fluorescein (1 mol %) in 1:1chloroform:methanol and subjecting the mixture to reverse phaseevaporation through several cycles of freeze-thaw, followed by extrusionthrough a 200 nm polycarbonate membrane to achieve unilamellar vesicles.The formulations of the liposomes used in the experiments are shown inTable 1. For singly-targeted liposomes, the DSPE-PEG-GSSSGRGDSPA (SEQ IDNO: 2) or DSPE-PEG-DAEWVDVS (SEQ ID NO: 5) concentration was kept at 5mol % total lipid, while for the dual-targeted liposomes theDSPE-PEG-peptide composition was kept at 2.5 mol % total lipid for eachtype of targeting peptide, such that the cumulative DSPE-PEG-peptidecomposition was still at 5 mol % total lipid. The size distribution ofthe liposomal constructs was characterized using dynamic lightscattering (DLS) and the constructs were found to have an averagediameter of w150 nm following extrusion (FIG. 2).

TABLE 1 Components of liposomes used in the experiments. LiposomeLiposome type components Non-targeted DSPC: 49 mol % DSPE-PEG: 5Cholesterol: 45 DSPE-PEG- (Unmodified) mol % mol % Rhodamine orFluorescein: 1 mol % GPIIb-llla- DSPC: 49 mol % DSPE-PEG- Cholesterol:45 DSPE-PEG- targeted (RGD- GSSSGRGDSPA mol % Rhodamine modified) (SEQID NO: 2): or Fluorescein: 1   5 mol % mol % P-selectin- DSPC: 49 mol %DSPE-PEG- Cholesterol: 45 DSPE-PEG- targeted DAEWVDVS mol % Rhodamine(DAEWVDVS- (SEQ ID NO: 5): or Fluorescein: 1 modified)   5 mol % mol %Dual-targeted DSPC: 49 mol % DSPE-PEG- Cholesterol: 45 DSPE-PEG-(modified GSSSGRGDSPA mol % Rhodamine simultaneously (SEQ ID NO: 2): orFluorescein: 1 with both 2.5 mol % mol % peptides) DSPE-PEG- DAEWVDVS(SEQ ID NO: 5): 2.5 mol %

In Vitro Studies to Confirm Targeting Specificity and Enhanced BindingStability of Constructs

Receptor-specific binding studies in a static environment human wholeblood was obtained from healthy, aspirin-refraining donors byvenipuncture following IRB-approved protocol and collected in citratedtubes. Platelet-rich-plasma (PRP) was obtained from the whole blood bycentrifugation (150×g, 15 min, room temperature), and plateletconcentration was verified at 200×10⁶ platelets/ml. The platelets in PRPwere activated using ADP at a concentration of 2×10⁻⁵M, and were allowedto adhere to glass coverslips pre-coated with collagen. The adheredplatelets were subsequently fixed with 4% paraformaldehyde, and theirpresence on the coverslip was confirmed by immunostaining withAlexaFluor 647-anti-CD62P (staining P-selectin) and FITC-anti-CD41a(staining integrin αIIbβ3) followed by fluorescence microscopy imaging.For establishing the receptor specificity of liposome binding,collagen-coated coverslip adhered platelets were incubated withfluorescently-labeled, non-targeted (unmodified) or αIIbβ3-targeted(RGD-modified) or P-selectin-targeted (DAEWVDVS (SEQ ID NO:5)-modified)liposomes (schematics in FIGS. 3 and 4). After 1 h of incubation, thecoverslips were gently washed with PBS to remove loosely bound liposomalconstructs and imaged using fluorescence microscopy. All images werecaptured using a Zeiss Axio Observer. D1 inverted fluorescencemicroscope fitted with a photometrics chilled CCD camera and a 63×objective using exposure time of 800 ms. To confirm that non-specificbinding of the liposomes was not occurring, fluorescently-labeledpeptide-modified liposomal constructs were incubated with coverslipscoated with collagen only but no activated platelets, followed by gentlewashing and imaged using fluorescence microscopy keeping same exposuretimes (800 ms).

Receptor-Specific Blocking Studies in a Static Environment

As additional verification of the peptide-modified liposomal constructsbinding specificity to the respective activated platelet surface targetreceptors, blocking studies were carried out. First, activated plateletswere adhered onto collagen coated coverslips as previously described.Next, nonfluorescent RGD-modified or DAEWVDVS (SEQ ID NO:5)-modifiedliposomal constructs were incubated with the coverslip adhered plateletsfor 1 h. After 1 h, the coverslips were gently washed with PBS andfurther incubated with fluorescent FITC anti-CD41a or AlexaFluor647-anti-CD62P for 30 min. Similar activated platelets adhered ontocollagen-coated coverslips were incubated with FITC-anti-CD41a orAlexaFluor 647-anti-CD62P without prior incubation with thenon-fluorescent peptide-modified liposomes. The rationale was that ifthe peptide-modified liposomal constructs bound their specific targetreceptors, then the pre-incubation with the liposomes would possiblyoccupy many of these receptors and block them from binding thefluorescent antibodies in the subsequent step (schematics in FIGS. 5 and6). On the other hand, without liposome pre-incubation, the fluorescentantibodies would successfully stain the respective receptors. Allcoverslips were imaged as previously described.

Flow Cytometry Analysis of Platelet-Targeting by Peptide-ModifiedConstructs

Liposomal construct binding to activated platelets was also studiedusing an LSRII (Becton Dickinson) flow cytometer (Blue 488 nm and Red640 nm lasers). Whole blood aliquots were incubated with ADP for 30 minto activate the platelet population. Samples were then fixed with 4%paraformaldehyde for 1 h. The level of platelet activation was assessedby co-staining the aliquot with FITC-conjugated anti-CD41a antibody andAlexaFluor 647-conjugated anti-CD62P antibody and running the samplethrough the flow cytometer to assess the fluorescence associated withthe gated platelet population. Following confirmation of plateletactivation, duplicate whole blood aliquots were incubated with RhodamineB-labeled non-targeted (unmodified), singly-targeted (RGD-modified ORDAEWVDVS (SEQ ID NO:5)-modified), or dual-targeted (both RGD andDAEWVDVS (SEQ ID NO:5)-modified) liposomes at a final concentration of500 mM total lipid for 30 min and run through the flow cytometer toanalyze platelet-associated fluorescence. For control studies, thealiquots were subjected to liposome incubation without pre-incubationwith ADP (no platelet activation). For all analyses, gated plateletpopulation fluorescence was analyzed at 20,000 counts per aliquot.

Binding Stability and Retention Studies in Parallel Plate Flow Chamber(PPFC)

A standard PPFC system (10 mm width-0.01 in height) fitted to aperistaltic pump and placed under an inverted epifluorescence microscopewas used for studying interaction of liposomal constructs withsurface-adhered platelets under a flow environment (schematic in FIG.7). The PPFC system allows variation of wall shear stress (T_(w)) at theplate surface by modulating the flow rate (Q) of the fluid through thechamber, according to the equation τ_(w)=6 μQw⁻¹ h⁻², where ‘μ’ is fluidviscosity, ‘w’ is chamber width and ‘h’ is chamber height. Similar flowchamber set-ups have been reported for analysis of cell-materialinteractions in a dynamic flow environment. For PPFC studies, glassmicroscope slides were coated in two equal circular area regions withalbumin and collagen. The slides were allowed to sit in contact with PRPin presence of ADP or BSA for 1 h to render adhesion of activatedplatelets to the collagen coated region; the albumin-coated region doesnot adhere activate platelets and hence act as the control area. Afterfixing with 4% paraformaldehyde and immunostaining with FITC-anti CD41aand AlexaFluor 647-anti-CD62P, the collagen-coated region revealed denseadhesion and aggregation of platelets (simulating a thrombotic region),while the albumin coated region had almost no adhered platelets. Thisfluorescence microscopy information was further complimented by ScanningElectron Microscopy (SEM) images (representative images shown in FIG.7). After confirming the activated platelet-adhered andplatelet-deficient regions on the glass slides, similar PRP-incubatedslides but without platelet immunostaining were placed in the PPFC, andRhodamine-labeled (red fluorescent) peptide-modified (singly-targeted ordual-targeted) or unmodified liposomes were allowed to flow through thechamber in a PBS suspension (total lipid concentration of 1 mM) for 30min. The flow was maintained at various flow rates for different batchesof experiments to allow varying wall shear stress values in the rangebetween 5 and 60 dyn/cm². After 30 min, the liposome solution wasreplaced with PBS and the flow was maintained for another 15 min todetermine the stability of binding of the liposomes in the dynamic flowenvironment. Throughout this procedure, at various time points and forvarious flow rates (hence shear stresses), the glass slides were imagedunder an epifluorescence microscope as previously described.

Data Analysis

For fluorescence images from the binding/blocking studies under bothstatic and dynamic conditions, adhesion and retention was quantifiedusing raw image analysis of surface averaged intensity values in AdobePhotoshop CS4 software. Statistical analysis of the staticbinding/blocking results was done using Paired Student's t-tests, andstatistical analysis of dynamic binding results was done using ANOVA.For all statistical analysis, significance was considered at p<0.05.

Results

We have developed liposomal nanoconstructs that are simultaneouslysurface modified with two types of peptide ligands having specificityand high affinity to two different cell-surface receptors on activatedplatelets, namely integrin αII_(b)β₃ (also known as glycoproteinGPIIb-IIIa) and P-selectin (FIG. 1).

Receptor-Specific Binding Studies and Blocking Studies in a StaticEnvironment

FIGS. 3 and 4 show representative fluorescence microscopy images for thereceptor-specific binding studies with RGD-modified and DAEWVDVS (SEQ IDNO:5)-modified liposomal constructs, respectively, along withcorresponding quantitative data of surface-averaged fluorescenceintensity analysis. As evident from the data, both RGD-modified andDAEWVDVS (SEQ ID NO:5)-modified liposomal constructs were able tosignificantly bind activated platelets by specific interaction withtheir respective target receptors, but had minimal non-specific bindingwith collagen itself in the absence of adhered active platelets.Constructs without any peptide modification or bearing negative controlpeptides had negligible binding to activated platelets. The targetreceptor specificity of the binding was further confirmed by the resultsof the receptor blocking studies as shown in FIGS. 5 and 6. As evidentfrom the figures, pre incubation with the RGD-modified nonfluorescentconstructs was able to block subsequent binding of FITC-anti-CD41a tointegrin αIIbβ, while pre-incubation with the DAEVWVDVS (SEQ IDNO:5)-modified nonfluorescent constructs was able to block subsequentbinding of AlexaFluor 647-anti-CD62P to P-selectin on activatedplatelets.

Flow Cytometry Analysis of Binding

FIG. 8 shows representative flow cytometry scatter plot of the wholeblood aliquot, the gated activated platelet population analyzed forfluorescence, and representative fluorescence histograms ofADP-activated aliquots incubated with non-targeted, singly-targeted(RGD-modified or DAEWVDVS (SEQ ID NO:5)-modified) and dual-targeted(simultaneously RGD- and DAEWVDVS (SEQ ID NO:5)-modified) liposomalconstructs. As evident from the data, the RGD-modified and the DAEWVDVS(SEQ ID NO:5)-modified constructs were individually able to bindactivated platelets significantly higher than non-targeted constructs.Furthermore, when the two peptide modifications were combined on asingle construct, the resultant dual-targeted constructs showed muchenhanced binding to activated platelets compared to the singly-targetedconstructs. This validates our rationale that the dual targetingapproach will render enhanced targeting efficacy toward activatedplatelets, which in essence, would enhance the binding selectivity ofthese constructs at sites of vascular disease where large numbers ofactivated platelets are involved.

Binding Stability and Retention Studies in Parallel Plate Flow Chamber(PPFC)

FIG. 9 shows representative fluorescent images of construct interactionwith test (platelet-covered) and control (albumin without platelet)surface regions on the glass slide in PPFC at three shear values (5, 25and 45 dyn/cm2) for the 30 min time point. The bottom panel of FIG. 9also shows quantitative analysis of surface-averaged fluorescence fromimages at three shear stress values over a period of 45 min (30 min ofliposomal construct flow in recirculating loop+15 min of plain PBS flowin open loop) for the various test and control samples. As evident fromthe qualitative images, the RGD-modified liposomal constructs, theDAEWVDVS (SEQ ID NO:5)-modified liposomal constructs, and thedual-targeted constructs were all able to significantly bind theactivated platelet covered surface under flow compared to unmodified(non-targeted) liposomal constructs or platelet-deficient (albumin)surfaces. The quantitative analysis shows that the dual-targetedliposomal constructs have significantly enhanced binding and retentionon activated platelets under flow compared to the singly-targetedconstructs. This validates our rationale that the dual targeting notonly enhances the selectivity of activated platelet targeting(complementary results to flow cytometry data), but also enhances thestrength of binding to ensure higher retention at the target site undera dynamic flow environment.

Nanovehicle Binding to Active Platelets Using Varying Mole % of LigandsDirected to GPIIb-IIIa and P-selectin

FIG. 10a-c shows that multi-receptor targeted strategy results in higherbinding and retention of nanovehicles on active platelets under flowconditions, compared to single receptor targeting at the same mol % andthat multi-receptor targeting allows for achieving enhanced binding andretention with lower mol % of total ligands, compared to single receptortargeting with only one type of ligand.

Nanovehicle Binding to Active Platelets Using Fixed Mole % but VaryingRatios of Ligands Directed to GPIIb-IIIa and P-Selectin

FIG. 11a-c show that varying the ratios of surface FMP (fibrinogenmimetic protein, the RGD motif) and SMP (selectin p ligand (SELPLG)mimetic protein) while maintaining a fixed mole % of total ligandsdirected to GPIIb-IIIa and P-selectin, allows for achieving enhancedbinding and retention to activated platelets under flow under a range offlow condition (5-55 dynes/cm²). FIG. 27 shows that multi-receptortargeted strategy results in higher binding and retention ofnanovehicles on active platelets under flow conditions, compared toplatelet-mimetic liposomes having varying ratios of von Willebrandfactor-binding peptides (VBPs) and collagen-binding peptides (CBPs).

Example 2 Affinity Manipulation of Surface-Conjugated RGD-Peptide toModulate Binding of Liposomes to Activated Platelets

We postulated that modification of liposome surface withGPIIb-IIIa-specific RGD motifs will result in enhancement of plateletaffinity, when compared with linear RGD (lRGD) motifs. The cyclic RGDpeptide, CNPRGDY(OEt)RC (SEQ ID NO:3) (terminal cysteines (C) cyclizedthrough disulfide), has been reported to have high affinity andselectivity for GPIIb-IIIa (α_(IIb)β₃), compared with otherRGD-recognizing receptors like αvβ₃, αvβ₅ sand α₅β₁. Hence this cyclicRGD peptide (cRGD) was developed by solid phase synthesis and conjugatedto lipid for incorporation into liposomes, such that the peptide ligandsstay displayed on the liposome surface. FIG. 16 shows a schematic modelof liposomes surface-modified by fibrinogen-mimetic cRGD-peptide foractive platelet-selective thrombus-targeted delivery. Liposomessurface-modified with moderate affinity linear RGD and non-specificlinear RGE motifs were used as comparison controls. The interaction ofthe surface-modified liposomes with activated platelets was studied invitro by microscopy and flow cytometry and ex vivo by microscopy.

Reagents and Supplies

All amino acid derivatives and peptide synthesis reagents were purchasedfrom Anaspec Inc. All lipids were purchased from Avanti Polar Lipids andNOF America Corp. For fluorescence studies1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzodiazol-4-yl)]amino]dodecanoyl]-sn-glycero-3-phosphocholine(PDPC-NBD, emission 534 nm, green fluorescence) was purchased fromAvanti Polar Lipids and, AlexaFluor-546-tagged human fibrinogen(AlexaFluor-546-Fg, emission 573 nm, red-orange fluorescence) waspurchased from Invitrogen and stored at −20° C. prior to use.Cholesterol, bovine serum albumin (BSA), phosphate buffered saline (PBS)and sodium citrate were obtained from Sigma Aldrich. Mass spectrometryand microscopy supplies were used from stock at respective facilities atCase Western Reserve University.

Development of Peptides and Peptide-Lipid Conjugates

An 11-residue linear RGD-containing peptide (GSSSGRGDSPA(SEQ ID NO: 2),lRGD), a 9-residue linear precursor of cRGD peptide (CNPRGDY(OEt)RC (SEQID NO:3) and an 11-residue RGE-containing negative control peptide(GSSSGRGESPA (SEQ ID NO:7), lRGE) were synthesized using standard FMocchemistry by solid-phase peptide synthesizer (ABI433A, AppliedBiosystems). For cRGD synthesis, the terminal cysteine residues of thelinear precursor were cyclized by a disulfide bond usingferricyanide-mediated oxidation process. All peptides were purified byHPLC and chemical purity was confirmed by mass spectrometry (MALDI-TOF).Peptide-lipid conjugates were prepared following using known methods.For this, peptides, while still on the resin, were reacted through theirN-terminal, to an N-hydroxysuccinimide (NHS)-activated polyethyleneglycol carboxyester derivative of distearoylphosphatidylethanolamine(DSPE-PEG-COO—NHS, from NOF America Corporation) and the resultantlipid-peptide conjugates were then cleaved from the resin, purified bydialysis and characterized by mass spectrometry. For lipid-cRGDconjugate, the linear peptide precursor was conjugated to lipid firstand the cyclization via disulphide bond was performed subsequently.

Platelet-Affinity of Free Peptides

Affinity of free lRGD and cRGD peptides to bind activated platelets wascharacterized by ‘half maximal Inhibitory Concentration’ (IC₅₀) value,which, in our assays, was the concentration of peptide required toinhibit fibrinogen-mediated platelet aggregation in platelet-rich plasma(PRP) by 50%, in an aggregometry (Bio/Data, PAP-4) set-up. PRP wasprepared from citrated human whole blood by centrifugation at 800 RPMfor 15 min at 25° C. 50 μl aliquots of PRP were warmed to 37° C. for 2minutes, incubated with various concentrations of lRGD and cRGD peptidein the presence of agonist adenosine di-phosphate (ADP, 1 μM) and themaximal aggregation percentage was determined while stirring for 15 min.Consequently, from the maximal aggregation percentage and comparison toaggregation without peptides, inhibition percentage was determined.

Preparation of Peptide-Modified Liposomes

All liposomes were prepared by reverse-phase evaporation followed byextrusion through nanoporous (100 nm) Nuclepore polycarbonate membrane,as described previously for our lRGD-liposomes. In all formulations, thefinal peptide-lipid conjugate content was kept at 1 mol %, which islower than our previously reported lRGD-liposome formulations with 5 mol% peptide-lipid conjugate. The rationale was that if liposomesurface-modification with a higher affinity peptide (e.g., cRGD) has anenhancing effect on platelet binding, the enhancement will be moresensitive and discernible at low peptide concentrations. PDPC-NBD wasincorporated at 1 mol % in the formulations as a fluorescent probe.Liposome size and stability were characterized and monitored by dynamiclight scattering using a Model 90Plus Brookhaven Instruments CorpParticle Size Analyzer for 30 days from the day of liposome preparation.

Microscopy Analysis of Liposome Binding to Platelets In Vitro

Monolayers of platelets were adsorbed from human platelet suspensiononto collagen III-coated glass coverslips following similar methods aswe have previously described for our lRGD-liposome studies. 3 μl of ADP(1 μM) in 120 μl PBS was added onto the platelet-adhered coverslips toensure sufficient activation of adhered platelets. The presence ofsurface-adsorbed platelets was confirmed by staining with fluoresceinisothiocyanate (FITC)-tagged anti-GPIIb-IIIa monoclonal antibody(FITC-anti-CD41a mAb, from BD BioSciences) and observing with a NikonDiaphot epifluorescence microscope. The ‘activated’ state of theadherent platelets was confirmed by scanning electron microscopy (SEM)of coverslip samples. For this purpose, the coverslip-adhered plateletswere fixed in 2.5% glutaraldehyde for 2 hours at 4° C., subjected toprogressive dehydration with graded series of ethanol solutions andfinally critical point dried in liquid CO₂. The SEM analysis was done byattaching the platelet-adhered coverslips to sample stubs and sputtercoating with platinum followed by observation with a Hitachi S-4500field emission electron microscope with an accelerating voltage of 5 kV.

For fluorescent liposome binding studies, coverslip-adhered plateletswere co-incubated with PDPC-NBD labeled lRGD, cRGD or lRGE-modifiedliposomes (diluted to a final concentration of 2.5 μM) andAlexaFluor-546-Fg in presence of 5 mM CaCl₂), for 1 hr at roomtemperature in the dark. The [fibrinogen: liposome] ratio in theincubation assays was maintained at 400:1 by mole, to provide aphysiologically relevant competitive environment. Subsequently thecoverslips were washed with PBS, fixed in 1% paraformaldehyde (PFA) for30 min at 37° C., and mounted on glass slides to be imaged by theepifluorescence microscope for simultaneous detection of NBD andAlexaFluor-546 fluorescence. A 40× oil-immersion objective was used tovisualize fluorescence from platelets. Images were collected inMetaMorph™ (Universal Imaging Corp.), using an exposure time of 500 msand intensity analysis was performed in the software accordingly.Fluorescence intensity was recorded as mean intensity per pixel area forstatistical analysis. All coverslips were prepared in duplicate, and atleast three fields were examined for each well.

Flow Cytometry Analysis of Liposome Binding to Platelets In Vitro

The effect of platelet activation on liposome binding was studied by aFACScan flow cytometer (Becton Dickinson) with a 488 nm laser and threecolor detector. For cytometry analysis whole blood aliquots withdifferent platelet activation levels were used. The level ofplatelet-activation was assessed by simultaneous fluorescence stainingof platelet integrin GPIIb-IIIa (with FITC-anti-CD41a) and P-Selectin(with PE-anti-CD62P). Previously we have reported on detectingprogressive activation of platelets in freshly drawn blood with additionof agonists (e.g., ADP) and have confirmed that binding of RGD-modifiedliposomes to platelets increased in an activation-dependant manner.Following a similar protocol, whole blood aliquots containingpredominantly activated platelets were incubated with PDPC-NBD-labeledcRGD-, lRGD-, or lRGE-modified liposomes at a final concentration of 50μM total lipid. For the liposome incubated samples, FITC-anti-CD41alabeling was not used, while PE-anti-CD62P labeling was still used as anactivated platelet marker. Simultaneous PE and NBD fluorescence, forgated platelet populations, was analyzed for ˜20,000 events (counts) persample aliquot and fluorescence data were recorded accordingly.

In Vivo Interaction of Surface-Modified Liposomes with ActivatedPlatelets and Ex Vivo Imaging and Analysis

For our in vivo model, an acute vascular injury was created by a ballooncatheter-induced endothelial denudation of the luminal wall in the leftcommon carotid of Lewis rats. After induction of anesthesia with anintraperitoneal injection of xylazine and ketamine cocktail, a midlinecervical incision was made to expose the left external carotid artery.The external carotid artery was ligated, and the internal carotid arterywas ligated temporarily. A 2F Fogarty balloon catheter (BaxterHealthcare Corp) was introduced through the arteriotomy site of theexternal carotid artery. The catheter was passed into the aortic arch,and the balloon was distended with saline until a slight frictionalresistance was felt on traction. The catheter was withdrawn through theleft common carotid with a slight rotational movement to ensureendothelial denudation of the luminal wall. The insertion and retractionprocedure was repeated three times to induce sufficient vascular injuryand hence an acute thrombotic environment. Subsequently, blood perfusionthrough the injury site was re-established by releasing the temporaryligation and maintained for 1 hr to allow platelet adhesion, activationand aggregation. To ascertain the presence of activated thromboticplatelets, two animals were sacrificed, their injured carotid vesselsections excised and after careful sample preparation, imaged with SEM.After the 1 hr blood reperfusion period, 25 μl of NBD-labeled cRGD-,lRGD- or lRGE-liposome suspension was injected into the ascending aortaproximal to the injury site. All vessels branching from the aorta,except the injured carotid artery, were ligated to maximize the flow ofliposomes along with the blood through the injury site. The blood (henceliposome) flow was maintained for 15 min, following which, the rats wereeuthanized, and the injured vessel was gently flushed with salinethrough the aorta, to remove artifacts. The injured artery section wasexcised, and immersed in 4% PFA solution for fixing. The fixed arterysection was then cut open longitudinally and mounted on glass slideswith the luminal injured liposome-exposed wall facing up. The exposedwall was imaged with a fluorescence microscope for NBD fluorescence.

Data Analysis

Statistical analysis, where applicable, was performed in MicrosoftExcel. A two tailed, unpaired student's t test (assuming unequalvariance) was performed to compare the difference in fluorescenceintensity of platelets stained by test and control liposomes in the invitro and in vivo assays. Significance was considered as p<0.05. Alldata was noted as mean±SD.

Results Development of Peptides and Lipid-Peptide Conjugates

FIG. 17 shows the peptide sequence structure and representative massspectrometry data of the (17 a) linear and (17 b) cyclic RGD peptidesdeveloped for targeting GPIIb-IIIa. These peptides were conjugated tolipid (DSPE-PEO-NHS) by amide bond formation between amino terminal ofpeptide and activated carboxyl terminal of lipid-PEG. FIG. 17c shows arepresentative MALDI-TOF analysis data of the product after conjugationof the cRGD peptide to DSPE-PEG-COO—NHS on resin, followed by cleavage.As evident from 17c, the mass spectrometry results of the crude productfrom the lipid-peptide conjugation reaction followed by cleavage fromresin, showed presence of residual unconjugated peptide and a broad‘hedgehog’ peak for the conjugated lipopeptide. The ‘hedgehog’appearance of the peak is attributed to the polydispersity of the PEGspacer block in the commercial DSPE-PEG-COO—NHS. The lipopeptide productwas purified from unconjugated free peptide by HPLC and dialysis.

Verification of Higher Affinity of the cRGD Peptide

FIG. 18 shows the effect of free peptide concentration (lRGD and cRGD)on the inhibition of platelet aggregation, as studied by aggregometryassays. As evident from the result, free cRGD causes 50% inhibition ofplatelet aggregation (dotted line in FIG. 17) at a much lower peptideconcentration compared to free lRGD. ‘Affinity’ is a kinetic parameter,while ‘specificity’ is a thermodynamic parameter. In our assays, boththe GPIIb-IIIa-specific peptides (lRGD and cRGD) try to kineticallycompete with natural ligand fibrinogen in binding active plateletGPIIb-IIIa and hence prevent fibrinogen-mediated platelet aggregation.The higher affinity peptide can outcompete fibrinogen (therefore cause50% inhibition of platelet aggregation) at low peptide concentrationswhile the lower affinity peptide requires much higher concentration togain the kinetic advantage. Hence ‘affinity’ is directly correlated withthe IC₅₀ value and the value for cRGD was found to be about 1000 timeslower than that for lRGD. The significantly higher affinity of cRGD wasthus established.

Dynamic Light Scattering Analysis of Liposome Size Distribution andStability

The effective diameter of liposomes in various batches was found to be˜150 nm, when measured fresh after extrusion. The liposome suspensionswere stored in vials at 4° C. and the size distribution was monitoredfor 30 days using dynamic light scattering. No significant variation ineffective diameter of liposomes was found for the 30 day periodindicating stable non-aggregating vesicles.

Microscopy Studies of Platelet-Liposome Interaction In Vitro

In order to validate our postulation that surface-modification ofliposomes with higher affinity peptide provides a way to enhanceliposome binding to activated platelets in a physiologically relevantcompetitive environment, platelet-liposome interaction was studied invitro, where liposomes and fibrinogen were allowed to co-incubate withcollagen-III adhered activated platelets. The presence ofsurface-adhered active platelets was confirmed by SEM, as shown in FIG.19A. The platelets co-incubated with green fluorescent liposomes andred-orange fluorescent Fg, showed dual fluorescence due to simultaneousbinding. The Fg fluorescence was similar for samples co-incubated withlRGD- or cRGD-liposomes (FIG. 19. B1 and C1). However, plateletsco-incubated with cRGD-liposomes (FIG. 19. C2) showed significantlygreater (p<0.005) NBD (green) fluorescence compared to plateletsco-incubated with lRGD-liposomes (FIG. 19. B2). The mean NBDfluorescence intensity per pixel area of platelets incubated withcRGD-liposomes was found to be significantly higher (FIG. 19. D)compared with platelets incubated with the lRGD-liposomes, fortriplicate batches. This indicates that in a competitive environmentcRGD-liposomes bind activated platelets at levels significantly higherthan lRGD-liposomes.

Flow Cytometry Studies of Platelet-Liposome Interaction In Vitro

The microscopy results were complimented by flow cytometry assays, wherewhole blood aliquots were incubated with NBD-labeled peptide-modifiedliposomes containing 1 mol % lipopeptide by composition. Freshly drawnblood showed only about 25% activated platelets whereas agonist-added(e.g., ADP) blood showed about 99% of the platelets to be activated, asconfirmed by co-staining with GPIIb-IIIa-specific andP-selectin-specific markers (FIGS. 20. A and B). Blood aliquotscontaining predominantly resting platelets showed a minimal level of NBDfluorescence staining of platelets over background (unlabeled) whenincubated with lRGD, cRGD or lRGE-liposomes (data not shown), suggestinga low degree of lipid exchange-based non-specific staining. However, foragonist-added blood aliquots, incubation with cRGD-liposomes resulted inincrease of platelet-associated NBD fluorescence by about an order ofmagnitude higher compared to that from 1-RGD liposome-incubated aliquots(FIG. 20, C). This confirms that liposome modification with higheraffinity cRGD peptide renders higher platelet binding ability comparedto modification with moderate affinity lRGD peptide.

Platelet-Binding of RGD-Modified Liposomes In Vivo

SEM was used to confirm the presence of thrombotic activated plateletsat the vascular injury site of the rat carotid. FIGS. 21A and 21B showrepresentative SEM micrographs of the carotid artery wall in nativestate and after catheter-induced injury, respectively. Numerousactivated platelets are visible adhered to the injured vessel wall. FIG.21C is a representative SEM micrograph of an injured artery wall afterallowing blood flow for 1 hr, showing considerable thrombosis as evidentfrom the fibrin clot ‘mesh’. FIG. 21D, E, F and G show representativefluorescence microscopy images of the injured carotid section luminalsurface exposed to no liposomes and, lRGE-, lRGD- and cRGD-liposomes,respectively. From analysis of mean fluorescence intensity of imagesfrom three batches of experiments, it was found that the injured carotidartery exposed to cRGD-liposomes had significantly higher (p<0.01) NBDfluorescence than that exposed to lRGD-liposomes.

Example 3 In Vitro and In Vivo Platelet Targeting by Cyclic RGD-ModifiedLiposomes

We have developed liposomes whose surfaces are decorated with multiplecopies of GPIIb-IIIa specific RGD ligands such that they canspecifically bind activated platelets. Here, we report our recentmicroscopy studies of active platelet binding by liposomes surfacedecorated with a high affinity cyclic RGD peptide. To assessplatelet-binding ability, we incorporated a lipophilic fluorophorewithin the liposomes and incubated test (surface modified with specifictargeting cyclic RGD peptide) and control (surface modified withnon-specific RGE peptide) liposomes with activated human platelets,followed by analysis of the incubated cells with fluorescence microscopyand phase contrast microscopy. The resolution limits of fluorescencemicroscopy resolution makes it difficult to visualize nanoscaleliposomes on activated platelets. To obtain this complimentary evidence,we developed the same liposomes where instead of a fluorophore, weincorporated Nanogold® in the liposomal membrane, and analyzed theliposome-incubated platelets by high resolution scanning electronmicroscopy (SEM). After confirming platelet-targeting in vitro byfluorescence, phase contrast and SEM techniques, the liposomes weretested in vivo in rats in an acute restenosis model created bycatheter-induced endothelial denudation of carotid artery. Theliposome-exposed injured vessels were excised from euthanized animalsand were imaged ex vivo by fluorescence microscopy and SEM.

Materials and Methods Reagents

Amino acid derivatives, activator (1-hydroxy-1-azabenzotriazoleuronium,HATU), and synthesis resin were purchased from Anaspec, Inc. Anactivated, purified synthetic polyethylene oxide (PEO) derivative ofdistearoylphosphatidylethanolamine containing a terminalN-hydroxysuccinimide activated carboxyester (DSPE-PEG-COO—NHS) waspurchased from NOF America Corporation. The PEO component had a reportedaverage molecular weight of 2000. Cholesterol, distearoylphosphatidylcholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (abbreviated as DSPE-PEO2000) and1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzodiazol-4-yl)]amino]dodecanoyl]-sn-glycero-3-phosphocholine(abbreviated as PDPC-NBD) were purchased from Avanti Polar Lipids. Forthe synthesis of nanogold labeled liposomes, dipalmitoyl phophatidylethanolamine Nanogold® (abbreviated as DPPE-Nanogold®) was purchasedfrom Nanoprobes (Yaphank, N.Y.) and refrigerated at 2-8° C. Bovine serumalbumin (BSA), human collagen III, phosphate buffered saline (PBS), HPLCgrade solvents and sodium citrate were obtained from Sigma Aldrich (St.Louis, Mo.). For rat platelet staining, FITC-conjugated mouse anti-ratCD42d (staining rat platelet glycoprotein V) monoclonal antibody wasobtained from BD Biosciences. For in vitro platelet studies, human bloodwas obtained from aspirin-refraining consenting donors within thedepartment of Biomedical Engineering at Case Western Reserve University.

Synthesis of Peptide Ligands, Lipid-Peptide Conjugates andPeptide-Decorated Liposomes

The cyclic RGD-peptide, namely, CNPRGDY(-OEt)RC (SEQ ID NO:3)(designated as c-RGD, with terminal Cysteines cyclized), and a negativecontrol peptide GSSSGRGESPA (SEQ ID NO:7) (designated as RGE) weresynthesized by solid-phase peptide synthesis using standard9-fluoromethoxycarbonyl (Fmoc) chemistry. The rationale for choosingthis particular cyclic RGD-peptide sequence is based on IC₅₀ values ofcyclic peptides in GPIIb-IIIa binding assays reported in literature. Thedetails of the peptide synthesis and the subsequentcarbodiimide-mediated conjugation of amine-terminated peptide toN-hydroxysuccimimide (NHS)-activated carboxyl terminal of DSPE-PEG-COOH,followed by cleavage of this lipid-peptide conjugate from the resin,purification (by HPLC and dialysis) and characterization (by MALDI-TOFmass spectrometry), and subsequent formulation into liposomes usingreverse phase evaporation and extrusion methods, have been describedelsewhere. In the liposome formulations, the concentration of test orcontrol peptide-lipid conjugate was kept as 5 mol % of total lipid. Therest of the formulation consisted of 50 mol % DSPC, 44 mol %cholesterol, and, 1 mol % PDPC-NBD or DPPE-Nanogold. Liposomessurface-decorated with cyclic RGD peptide will be denoted ascRGD-liposomes and those decorated with RGE peptide will be denoted asRGE-liposomes henceforth. Dynamic light scattering (DLS) studies with aBrookhaven Model 90 Plus Particle Size Analyzer showed the averageliposome size to be around 150 nm for all batches. FIG. 22 shows aschematic of liposome preparation and representative DLS data forliposomes before and after extrusion.

Fluorescence and Phase Contrast Microscopy Studies and Flow CytometryStudies In Vitro

Collagen-coated glass coverslips bearing monolayer of human platelets,activated by agonist ADP, were prepared as described elsewhere. Adhesionof activated platelet monolayer on coverslips was confirmed by stainingrepresentative coverslips with fluorescein isothiocyanate (FITC)-taggedmouse anti-human CD41a monoclonal antibody that stains plateletGPIIb-IIIa, and observing the fluorescence (λemission˜525 nm, greenfluorescence) using a Nikon Diaphot inverted microscope containing afilter cube with a 450-490 nm excitation filter, a 510 dichroic mirrorand a 520-560 nm bandpass filter. After confirming presence ofplatelets, similar platelet-adhered coverslips were incubated with testcRGDliposomes or control RGE-liposomes (8 μl with 2 μM total lipid percoverslip) containing PDPC-NBD as the fluorescent label. The liposomeincubation was done in presence of 5 mM CaCl₂) solution since theinteraction of RGD to active platelet GPIIb-IIIa is facilitated by Ca++ions. The incubation was maintained for 1 hr in the dark. Afterwards thecoverslips were gently washed with PBS, mounted on glass slides andimaged by the fluorescence microscope using the same filter parametersas that for FITC fluorescence.

Parallel phase contrast image of the same field of view was obtained onthe same microscope, by shutting off the fluorescence laser, turning onthe phase contrast light source and changing the objective fromfluorescence to a phase contrast one, at same magnification. Toestablish that the specific binding of cRGD-liposomes is predominantlyto activated platelets compared to quiescent platelets, we performedflow cytometry assays similar to that described in previous publicationsfrom our group. Briefly, NBD-labeled cRGD liposomes were incubated withaliquots of freshly drawn human whole blood with or without the additionof agonist ADP. We have previously shown 17 by flow cytometric analysisof GPIIb-IIIa and P-selectin co-expression on the platelet surface, thatfreshly drawn human blood has about 25% platelet population in activatedstate due to procedural effects (blood draw, contact with syringe,contact with storage tube etc.). Upon addition of agonist (ADP or TRAP),this activation level increases to about 98%. NBD-labeled cRGD liposomeswere incubated at a concentration of 50 μM total lipid with 10 μl ofhuman whole blood aliquot without pre-incubation or with pre-incubationof ADP. Following 30 min of liposome incubation, the aliquots wereanalyzed in a Becton-Dickinson FACScan flow cytometer for NBDfluorescence. All aliquots were measured in triplicate and approximately15000 counts were recorded per aliquot. Aliquots without any liposomeincubation were used as ‘unlabeled’ controls. The NBD fluorescenceintensity from aliquots without liposome incubation, aliquots withliposome incubation but without ADP pre-incubation, and aliquots withliposome incubation after ADP pre-incubation, were recorded ashistograms and the plots were overlaid for comparison.

SEM Studies In Vitro

Platelets were adhered from suspension onto collagen III-coatedcoverslips in presence of ADP as described previously. Presence ofactivated platelets adhered onto the collagencoated coverslips werefirst confirmed by SEM to obtain results complimentary to thefluorescence studies. For this, the platelet-incubated coverslips werefixed in 2.5% glutaraldehyde for 2 hours at 4° C., washed with PBS,dehydrated with graded series of ethanol solution (10, 30, 50, 70, 90,95, and 100%) and critical point dried in liquid CO2. The SEM imagingwas done by attaching the coverslips to sample stubs and sputter coatingwith platinum followed by observation with a Hitachi S-4500 fieldemission scope. After confirmation of the presence of activated adheredplatelets, similar samples were incubated with Nanogold-incorporatedtest (cRGD) and control (RGE) liposomes for 1 hour at room temperature,in presence of CaCl₂). Liposome-incubated coverslips were again washedwith PBS to remove liposome suspension, fixed in 2.5% glutaraldehyde for2 hours at 4° C., rewashed with PBS, dehydrated with graded series ofethanol solution, critical point dried in liquid CO2 and analyzed withSEM using same instrument as described above. The microscope wasoperated in both secondary and back-scatter electron detection modeswith an accelerating voltage of 5 kV. All images were saved directlywith a digital image acquisition system (Quartz PCI).

Ex Vivo Microscopy Studies on Platelet-Targeting of Liposomes in a RatCarotid Injury Model

The catheter-induced acute injury model in rat carotid has beendescribed elsewhere. This procedure of carotid injury has been utilizedin several studies regarding role of platelets and other cells andbiomolecules in regulating restenotic and intimal events. Followinginjury, the right common carotid and the descending aorta were ligated.The blood flow was reestablished (repurfusion) to allow flow of bloodonly through the injured left carotid vessel. The blood flow was resumedfor 1 hr or 2 hrs to allow activation, adhesion and aggregation ofplatelets at the injury site due to an acute thrombotic/restenoticenvironment. Four animals, two immediately after injury and two after 1hr reperfusion, were sacrificed and the injured vessel sections wereexcised and washed gently in PBS. Uninjured carotid sections were alsoexcised from the already sacrificed animals for image analysis andcomparison purposes. All sections were cut open longitudinally to exposethe injured surface. One uninjured section and one 1 hr-repurfusedinjured section were subjected to incubation with FITC-tagged mouseanti-rat CD42d monoclonal antibody (emission˜525 nm, green fluorescence)which stains for rat platelet glycoprotein GPV 24. Following incubationfor 1 hr in the dark, the sections were fixed with 4% paraformaldehyde(PFA), washed gently with PBS, mounted on glass slides with luminalsurface exposed and imaged with epifluorescence microscope using thesame filter and objective specifications as used for the in vitrostudies. The other uninjured, injured and repurfused sections were fixedin 3.0% glutaraldehyde in PBS at 4° C. overnight, washed with 50 mMglycine in PBS to remove excess aldehydes, and then washed withdistilled water. Following this, the sections were dehydrated in agraded series of ethanol solutions as described previously for the invitro SEM samples, critical point dried in liquid CO2, mounted on samplestubs, sputter coated with platinum and observed with the previouslymentioned EM instrument in both secondary and back-scatter modes with anaccelerating voltage of 5 kV. The fluorescence and the SEM analyses ofthe injured carotid sections vs uninjured sections were done to confirmthe presence of activated adhered platelets in the acute injuryenvironment of our model. Following confirmation, similar animals wereexposed to fluorescently labeled or Nanogold-labeled test and controlliposomes. At the end of the 1 hr or 2 hr blood reperfusion periodthrough the injured vessel, NBD-labeled or Nanogold-labeled cRGD- andRGE. Liposomes were injected into the left ventricle of the rat heart toallow the flow of liposomes along with the blood pumped into the injuredcarotid. For the ex vivo imaging studies, statistical power calculationswere based on the area of coverage of the injured luminal surface byfluorescently labeled liposomes, as obtained by morphometric analysis(by Metamorph and Image J) of digital images. General background levelsappear to be ˜5% of the area. Our power calculation was based on seeinga 100% increase (to 10% coverage) with 80% power. Based on theseparameters our calculation suggested a sample size of ˜4 animals perliposome formulation. For NBD-labeled test and control liposomes, fouranimals were used per formulation. For Nanogold-labeled test and controlliposomes, two animals were used per formulation, since this analysiswas more qualitative. After liposome injection, the reperfusion flow wasmaintained for 15 minutes, at the end of which the rats were sacrificedand the injured vessels were excised, washed gently by PBS and preparedfor fluorescence or SEM imaging.

For fluorescence imaging, the sections were fixed in 4% PFA, re-washedwith PBS, sliced open longitudinally, and the luminal side was imagedwith the previously described inverted epifluorescence microscope forNBD fluorescence. At least eight images were obtained per section. ForSEM imaging, the excised tissue sections were sliced openlongitudinally, fixed in 3.0% glutaraldehyde, washed with 50 mM glycineand then in distilled water. To facilitate imaging of Nanogold-labeledliposomes within the complex morphology injured tissue, goldautometallography was used to enhance the size of Nanogold. This wasachieved using a commercially available autometallography kit“Goldenhance-EM” (Nanoprobes) and following the supplier instructions.The tissues were immersed in the enhancement solution for 15 minutes,which according to the supplier, can increase the Nanogold size to ˜20nm. Control tissue sections, without Nanogold-labeled liposome exposurebut with exposure to the Goldenhance solution were prepared to check fornon-specific background enhancement in SEM. After enhancement, allsections were washed with distilled water, dehydrated in a graded seriesof ethanol solutions, critical point dried in liquid CO₂ and analyzedwith SEM. At least eight images were obtained per tissue section.

Data Analysis

For in vitro and ex vivo fluorescence images of liposome-incubatedplatelets, surface averaged fluorescence intensity was measured usingMetamorph® software. For analysis and comparison purposes, the cameraparameters were kept constant for all in vitro studies. For the ex vivostudies, the camera conditions were different from the in vitroconditions, but were kept constant for all ex vivo images. Statisticalanalysis, where applicable, was performed in Microsoft Excel using a twotailed, unpaired student's t test (assuming unequal variance) to comparethe difference in fluorescence intensity. Significance was considered asp<0.05. All data was noted as mean±SD. For in vitro and ex vivo SEMstudies, the image acquisition parameters were kept constant and theresults were compared qualitatively.

Results In Vitro Fluorescence Microscopy Studies and Flow CytometryStudies on Liposome Incubated Human Platelets

FIG. 23 shows representative microscopy images for coverslip-adheredADP-activated human platelets incubated with (23A) RGE-liposomes and(23B) cRGD-liposomes, in presence of Ca++. FIGS. 23A and 23B arerepresentative of images captured through 1× objective. As evident fromthe images, qualitatively, the binding of cRGD-liposomes to theactivated platelets was considerably higher than that for RGE-liposomes.FIG. 23E shows the mean fluorescence intensity data for samplesincubated with RGE-liposomes and cRGD liposomes. Statistical analysis ofaverage fluorescence intensity of images from multiple batches ofexperiments showed that fluorescence intensity of NBD-labeledcRGD-liposome incubated platelet-adhered coverslips were significantlyhigher (p<0.01) compared to that from RGE-liposome incubatedplatelet-adhered coverslips, indicating enhanced platelet binding of thecRGD-liposomes. The cRGD-liposome incubated samples were observedfurther at higher magnification with 60× oil-immersion objective. FIGS.23C and 23D show representative fluorescence and phase contrast imagesrespectively, with 60× objectives, of the same field of view for ac-RGD-liposome incubated platelet-adhered coverslip. The one-to-onecorrespondence of the fluorescence spots with activated platelets can beobserved from 23C and 23D. Representative NBD fluorescence histogramsfrom flow cytometry analyses are shown in FIG. 23F; liposome incubationwith predominantly inactive platelet population (no ADP pre-incubation)resulted in NBD intensity close to that of baseline ‘unlabeled’ whilethat with ADP-activated platelets increased NBD fluorescence from thegated platelet population by almost two orders of magnitude. Suchanalyses further confirmed that the binding of the cRGD-liposomes werespecifically enhanced to active platelets, and the non-specific plateletbinding of cRGD-liposomes was minimal.

In Vitro SEM Studies on Liposome-Incubated Human Platelets

FIG. 24 shows representative images from SEM studies of platelet-adheredcoverslips incubated with Nanogold-labeled cRGD- and RGE-liposomes. FIG.24A and the enlarged image in FIG. 24B, are representative SEM images ofthe coverslip surface after the experimental procedure of incubating asuspension of human platelets on the collagencoated glass coverslips inpresence of agonist ADP. As evident from the images, the procedureresulted in formation of an adhered monolayer of highly activatedplatelets. The highly activated state is emphasized by the ‘spread’morphology, highly folded membrane appearance and the numerouspseudopodal extensions of the platelet membrane as seen in FIGS. 24A and24B. Similar coverslip-adhered platelets were incubated withNanogoldlabeled cRGD- and RGE-liposomes and imaged with SEM. Due to thehighly convoluted appearance of the activated platelet membrane, it isdifficult to conclusively distinguish between a membrane fold and avesicular liposome attached to the membrane from secondary electronimages. Hence, backscatter images were also obtained for the sameplatelets with the rationale that the gold-labeled nanoparticles, ifbound to the platelets, will produce enhanced backscatter and hence willenable higher resolution visualization of the nanoparticles, compared tothe normal backscatter from platelet membrane folds. FIGS. 24C1 and 24C2show respectively the secondary electron mode and the backscatter modeimages of a representative platelet on coverslips incubated withRGE-liposomes. While the secondary electron mode image shows numerousfolds and bulges on the membrane that apparently may look like liposomalparticles, the backscatter image of the same platelet shows only minimalcontrast-enhancement on the platelet membrane, thereby suggesting thatthe folds and bulges seen in the secondary electron mode image aremainly convolutions and vesicular protrusions of the platelet membraneitself (e.g., vesiculation into microparticles), with may be minimalnon-specific binding of gold-labeled liposomes by possible lipidexchange. FIGS. 24D1 and 24D2 show respectively the secondary electronmode and the backscatter mode images of a representative platelet oncoverslips incubated with cRGD-liposomes. Once again the secondaryelectron mode image shows folds, bulges and vesicular protrusions on theplatelet membrane similar to the RGE-liposome incubated samples.However, backscatter image of the same field shows significant brightcontrast of the platelet membrane compared to the representative samplefrom the RGE-liposome incubated batch. Also, multiple highlycontrast-enhanced vesicular structures (highlighted by arrowheads)adhered onto the platelet membrane and the platelet pseudopodalextensions are visible on the representative image from thecRGD-liposome incubated batch. From the scale bar, the size of thesestructures are around 100-200 nm and hence most possibly they areNanogold-labeled liposomes. These results suggest enhanced binding andfusion of goldlabeled cRGD-liposomes with the platelet membrane.Considering these results to be complimentary to the results obtainedfrom fluorescence microscopy studies, it can be concluded that thecRGD-liposomes have enhanced binding, specifically to activatedplatelets, and hence can be promising towards platelet-directedsite-selective vascular delivery.

Ex Vivo Microscopy Studies of Liposome-Platelet Interactions in RatCarotid Injury Model

FIG. 25 shows the schematic of experimental procedure (25A), acollection of representative SEM and fluorescent images of the carotidwall before (25B and 25D) and after injury (right after injury, 24E,and, after 1 hr repurfusion, 25C and 25F). FIG. 25 also showsrepresentative fluorescent images of the injured carotid exposed toNBD-labeled RGE-liposomes following 1 hr (25H) and 2 hr (25J)repurfusion, and the injured carotid exposed to NBD-labeled RGDliposomes following 1 hr (25I) and 2 hr (25K) repurfusion, along withthe statistical analysis of fluorescent images from all batches of thisstudy (25L). As evident from 25B, the uninjured rat tissue itself hasautofluorescence associated with it. The SEM image of the uninjuredtissue (25D) shows uniform striated appearance given by luminal liningof cells, while upon catheter-induced injury, acute disruption of theluminal wall is visible (25E). After perfusion of blood through theinjury site for 1 hr, staining with FTTC-tagged anti-rat CD42d andimaging with epifluorescence microscope revealed numerous adheredplatelets on the injured luminal wall (25C). This data was complimentedby SEM analyses of similar samples, where numerous adhered plateletswere visible on the injured wall (25F) and upon further magnification,platelets were found to be arrested in and adhered to fibrin mesh (25G),signifying an acute thrombotic/restenotic environment. The 15-minuteexposure of the injured carotid to NBD-labeled RGE-liposomes (in flowingblood) after repurfusion for 1 hr (25H) and 2 hrs (25J) showed onlyminimal staining of platelets over the background autofluorescence. Thesame for NBD-labeled c-RGD-liposomes showed significant plateletstaining over background autofluorescence for both 1 hr (25I) and 2 hr(25K) repurfusion situations. Statistical analysis of averagefluorescence intensity of images from multiple batches of experimentsconfirmed this observation (25L). As evident from the representativeimage 25K, fluorescence microscopy analysis of c-RGD-liposome exposedinjured carotid after 2 hr perfusion period also showed indication ofacute platelet aggregation and clotting (green fluorescent clumps in25K). FIG. 26 shows representative secondary mode and backscatter modeSEM images of injured carotid wall exposed to Nanogold-labeledRGE-liposomes (26A1 and 26A2) and cRGD-liposomes (26B1 and 26B2).Secondary electron mode images for both RGE-liposome and cRGD-liposomeexposed injured carotid wall looked very similar (26A1 and 26B1),revealing dense fibrin mesh and adhered/arrested platelets. However thebackscatter mode images for cRGD-liposome exposed injury site (26B2)showed considerably bright contrast compared to RGE-liposome exposedinjury site (26A2). This suggests enhanced binding of gold-labeledcRGD-liposomes to the injury site, most possibly, by design, to theactivated platelets adhered and aggregated at the site. Because of thehighly irregular three dimensional dense morphology of the thromboticenvironment, the dynamic flow environment of the experimental protocol,and the complex ex vivo sample preparation procedures, the intact vesselintegrity of platelet-adhered liposomes was probably not maintained inthese in vivo experiments, as it was maintained for the staticincubation experiments in vitro. Nonetheless, the enhanced brightness ofcRGD-liposome exposed tissue suggested enhanced site-specific liposomebinding, complimentary to the fluorescence microscopy data of FIG. 25.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents disclosed above are herein incorporated byreference in their entirety.

Having described the invention, we claim:
 1. A method of delivering atherapeutic and/or imaging agent to activated platelets in a subject,the method comprising administering to the subject a composition, thecomposition including a heteromultivalent nanoparticle or microparticlecomprising a therapeutic agent and/or imaging agent, the nanoparticle ormicroparticle having an outer surface and a plurality of targetingmoieties conjugated to the surface of the nanoparticle or microparticle,the targeting moieties including a first activated platelet targetingmoiety and a second activated platelet targeting moiety, wherein thecomposition is capable of binding activated platelets in the subjectunder a hemodynamic shear environment.
 2. The method of claim 1, thenanoparticle or microparticle construct comprising a liposome.
 3. Themethod of claim 1, the first activated platelet targeting moietycomprising a GPIIb-IIIa-binding peptide and the second activatedplatelet targeting moiety comprising a p-selectin binding peptide. 4.The method of claim 1, the GPIIb-IIIa-binding peptide comprising a RGDsmall peptide and the p-selectin binding peptide comprising a SEQ IDNO:
 1. 5. The method of claim 4, the RGD small peptide having SEQ IDNO:3.
 6. The method of claim 1, wherein the first and second activatedplatelet targeting moieties are conjugated to the nanoparticle ormicroparticle surface with PEG linkers.
 7. The method of claim 1,wherein the first and second activated platelet targeting moieties canbe spatially or topographically arranged on the nanoparticle ormicroparticle surface such that the first and second activated platelettargeting moieties do not spatially mask each other and the nanoparticleor microparticle is able to bind to an activated platelet with exposedactivated platelet receptors and enhance retention of the nanoparticleor microparticle onto activated platelets under hemodynamic flow.
 8. Themethod of claim 7, wherein the ratio of GPIIb-IIIa-binding peptide top-selectin binding peptide provided on the nanoparticle or microparticlesurface is about 80:20 to about 20:80.
 9. The method of claim 7, whereinthe GPIIb-IIIa-binding peptide and p-selectin binding peptide providedon the nanoparticle or microparticle surface have a total mol % of about5 to about 20 with respect to total lipid content.
 10. The method ofclaim 1, wherein the therapeutic agent is a thrombolytic agent and thesubject is afflicted with an occlusive vascular disease selected fromthe group consisting of stroke, myocardial infarction, peripheralarterial diseases and deep vein thrombosis.
 11. The method of claim 1,the nanoparticle or microparticle construct further comprising aplurality of Golden Nanorods (GNRs) conjugated to the surface, the GNRsallowing photothermal destabilization of the nanoparticle ormicroparticle construct and release of the therapeutic and/or imagingagent in response to near-infrared (NIR) light.